Nitrile hydratase and a method for producing amides

ABSTRACT

An objective of the present invention is to provide a nitrile hydratase capable of producing 2-hydroxy-4-methylthiobutyroamide. The present invention provides a novel nitrile hydratase producing α-hydroxyamide using, α-hydroxnitrile as the substrate, and the encoding DNA thereof. The enzyme can be obtained from  Rhodococcus  sp. Further, the enzymatic activity of the enzyme can he maintained stably during the reaction. The present invention provides a method for producing amide compounds, the method comprising the step of reacting this enzyme to nitrile compounds. According to the present invention, from hydroxy nitrile compounds, corresponding amide compounds can be produced biochemically without reducing the enzyme activity of nitrile hydratase.

TECHNICAL FIELD

The present invention relates to a novel nitrile hydratase and a methodfor producing amide compounds from nitrile compounds using the nitrilehydratase.

BACKGROUND ART

To improve the expression level of nitrile hydratase, methods thatoverexpress nitrile hydratase in microorganism cells by geneticengineering and that convert nitrile compounds to the correspondingamide compounds using the cells have been examined. For example, knownnitrile hydratases are derived form the following microorganisms. All ofthe nitrile hydratases are enzymes consisting of two types ofheterogeneous subunits.

The genus Rhodococcus (Examined Published Japanese Patent Application(JP-B) NO. Hei 3-54558)

The genus Rhodococcus (Unexamined Published Japanese Patent Application(JP-A) NO. Hei 2-119778)

The genus Pseudomonas (JP-A Hei 3-251184) The genus Rhodococcusrhodochrous (EP Patent Application NO. 455646)

However, in all cases, the expression level of nitrile hydrataseactivity is not high enough with E. coli transformed with an expressionplasmid containing an insert fragment of any nitrile hydratase genedescribed in these patent publications; the nitrile-hydrating activityper weight of cells of the transformant is lower than that of theoriginal microorganism from which the gene is derived (Ikehata, O.,Nishiyama, M., Horinouchi, S. and Beppu, T. “Primary structure ofnitrile hydratase deduced from the nucleotide sequence of a Rhodococcusspeices N-774 and its expression in Escherichia coli” Eur. J. Biochem.181(1989), 563-570; Nishiyama, M., Horinouchi, S., Kobayashi, M.,Nagasawa, T., Yamada, H. and Beppu, T. “Cloning and Characterization ofGenes Responsible for Metabolism of Nitrile Compounds from Pseudomonaschlororaphis B23” Journal of bacteriology 173 (1991):2465-2472;Kobayashi, M., Nishiyama, M., Nagasawa, T., Horinouchi, S., Beppu, T.and Yamada, H. “Cloning nucleotide sequence and expression inEscherichia coli of two cobalt-containing nitrile hydratase genes fromRhodococcus rhodochrous J1” Biochimica et Biophysica Acta. 1129(1991):23-33).

As mentioned above, it has been possible to express the nitrilehydratase activity itself in E. coli by genetic recombinationtechniques. However, so far, there is no available transformant havingso high nitrile hydration activity as to be used for the industrialproduction of amides.

On the other hand, a nitrile hydratase, which exhibits very high nitrilehydration activity, has been found in Achromobacter xerosis (IFO 12668),and the gene encoding the enzyme has been cloned. Further, the gene wasintroduced into E. coli using an expression plasmid, and nitrilehydratase was overproduced with the resulting transformant (JP-A Hei8-266277). This report shows only examples of acrylamide andα-hydroxyisobutylamide production, and thus there is no description onthe activity of this enzyme toward 2-hydroxy-4-methylthiobutyronitrilein the report.

Thus, little is known about nitrile hydratases that can use2-hydroxy-4-methylthiobutyronitrile (hereinafter abbreviated as HMBN) asa substrate. Furthermore, there is no previous method for producing2-hydroxy-4-methylthiobutylamides (hereinafter abbreviated as HMBAm)from 2-hydroxy-4-methylthiobutyronitrile using E. coli transformed withan expression plasmid containing the nitrile hydratase gene as an insertfragment.

On the other hand, with respect to the production of α-hydroxyamide bymicroorganism, there is a known method for producing the correspondingamides from lactonitrile, hydroxyacetonitrile, α-hydroxymethylthiobutyronitrile and such, using microorganisms belonging to thegenus Bacillus, the genus Bacteridium, the genus Micrococcus or thegenus Brevibacterium (see JP-B Sho 62-21519). In addition, there alsoexists a publicly known method for producing mandelamide fromcyanohydrin (see JP-A Hei 4-222591; JP-A Hei 8-89267).

However, the enzymes having the nitrile hydratase activity capable ofconverting nitrile compounds to amide compounds have a problem that theenzymes readily lose their own enzymatic activities due to the presenceof the nitrile compound as starting material or amide compound as theproduct. If the concentration of nitrile compound is raised in order toincrease the rate of amidation, the nitrile hydratase is readilyinactivated in a short period of time, and thus it is hard to obtainamide compound as the reaction product in a desired period of time. Inaddition, the amide compounds as the products also readily inactivatethe nitrile hydratase, and thus it is difficult to obtain a highconcentration of amide compound.

Furthermore, in varying degree depending on the type of compound,α-hydroxynitrile has been known to be partially decomposed to thecorresponding aldehyde and hydrocyanic acid in a polar solvent (see V.Okano et al., J. Am. Chem. Soc., Vol. 98, 4201 (1976)). In general,aldehydes are linked to proteins and can inactivate the enzymaticactivity (see Chemical Modification of Proteins, G. E. Means et al.,Holden-Day, 125(1971)). Further, like aldehyde, hydrocyanic acid(cyanide) can also inhibitorily act on many enzymes. Thus, aldehyde andcyanide produced from α-hydroxynitrile as the starting material can bethe cause of decreased enzymatic activity. Because of a problem that theenzyme is inactivated in a short period of time in the enzymatichydration or hydrolysis of α-hydroxy nitrile, it was difficult to obtaina high concentration of α-hydroxyamide with high productivity.

To prevent the loss of enzymatic activity, various methods forincreasing the enzymatic activity or for suppressing the loss ofenzymatic activity (inactivation) have been tested. Such attemptsinclude, for example, the following:

-   -   The reaction is carried out at a lower temperature ranging from        the freezing point to 15° C. (JP-B Sho 56-38118).    -   A lower concentration of substrate is continuously supplied from        multiple supply ports (JP-B Sho 57-1234).    -   A microorganism or processed product thereof is treated with an        organic solvent (JP-A Hei 5-308980).    -   The reaction is carried out in the presence of higher        unsaturated fatty acid (JP-A Hei 7-265090).    -   The microorganism cells are subjected to crosslinking treatment        with glutaraldehyde and such (JP-A Hei 7-265091; JP-A Hei        8-154691).    -   The concentration of hydrocyanic acid contaminated in the        nitrile compound is lowered by a chemical method, and then        nitrile hydratase is allowed to react with the nitrile compound        (see JP-A Hei 11-123098).    -   The long-term stabilization of the enzymatic activity is        achieved by the presence of sulfite ion, acid sulfite ion or        dithionite ion (see JP-A Hei 8-89267)    -   Aldehyde is added (see JP-A Hei 4-222591).

None of these methods had sufficient effects on the industrialapplications. Although some of the methods were effective, they had roomfor economical or practical improvement. For example, theabove-mentioned method adding aldehyde requires a large quantity ofaldehyde in 1-5 times molar excess of cyanohydrin as the start material,and thus the method was less than an economical solution. Similarly, itis illustrated that the method adding sulfite ion, acid sulfite ion ordithionite ion requires addition of the ion in an amount equivalent toor larger than that of the starting material, and thus the method wasnot practical one.

An objective of the present invention is to provide a nitrile hydratasehaving high nitrile-hydrating activity. Another objective of the presentinvention is to provide a stable nitrile hydratase capable ofmaintaining the high enzymatic activity over a long period of time. Inaddition, still another important objective of the present invention isto provide a nitrile hydratase capable of also using2-hydroxy-4-methylthiobutyronitrile as a substrate.

Furthermore, another objective of the present invention is to providethe gene encoding nitrile hydratase having high nitrile hydrationactivity, recombinant plasmid containing the gene and transformantcontaining the recombinant plasmid. In addition, yet another objectiveof the present invention is to provide a method for producing thecorresponding amides from nitrile using the transformant expressing highnitrile hydration activity.

DISCLOSURE OF THE INVENTION

The present inventors strenuously studied to achieve the above-mentionedobjectives, and found nitrile hydratase having very high nitrilehydration activity in microorganisms belonging to the genus Rhodococcus(Rhodococcus sp.). Then, the present inventors confirmed that thenitrile hydratase was a novel enzyme usable for achieving theabove-mentioned objectives, and thus completed the present invention.

The inventors then cloned the gene encoding the enzyme by recombinantDNA technology, and prepared E. coli transformed with the expressionplasmid comprising an expression vector containing the isolated gene asan insert. Further, the present inventors succeeded in the large-scaleproduction of the nitrile hydratase with the obtained transformant andthus completed the present invention.

Namely, the present invention provides the following nitrile hydratase,polynucleotide encoding the enzyme, method for producing the enzyme andmethod for producing amides using the enzyme of the present invention.

The nitrile hydratase of the present invention has the followingphysicochemical properties (a) and (b):

-   (a) acting on the nitrile group of nitrile compound, hydrating the    nitrile group and converting it to an amide group; and,-   (b) being cyanide-resistant.

In the present invention, the activity of hydrating the nitrile group ofnitrile compound and converting it to an amide group is referred to as“nitrile hydratase activity”. Preferably, an enzyme capable of acting onthe compound of the following formula (1) and producing the amidecompound of formula (3) is designated as “nitrile hydratase”.

Formula (1):

Formula (3):

(Where R represents substituted or unsubstituted alkyl group,substituted or unsubstituted alkenyl group, substituted or unsubstitutedcycloalkyl group, substituted or unsubstituted alkoxy group, substitutedor unsubstituted aryloxy group, substituted or unsubstituted saturatedor unsaturated heterocyclic group).

Preferably, the nitrile hydratase of the present invention can act on2-hydroxy-4-methylthiobutyronitrile and produce2-hydroxy-4-methylthiobutyroamide.

The nitrile hydratase activity of the present invention can be confirmedas follows. At first, an enzyme sample is added to a 0.1 M potassiumphosphate buffer (pH 6.5) containing 10% v/v2-hydroxy-4-methylthiobutyronitrile (HMBN) as the substrate. Instead ofthe enzyme sample, microorganism cells and crude enzyme are also usable.After addition of enzyme, the solution is incubated at 20° C. for 15minutes. The reaction solution is then combined with an excess volume of0.1% (v/v) phosphate solution, and the mixture is shaken vigorously tostop the reaction. The reaction product can be analyzed by HPLC.

According to this assay method, 1 U of nitrile hydratase was defined asthe amount of enzyme capable of producing 1 μmol nicotinamide at 20° C.for 1 minute in a reaction solution with a standard composition; 1 U wasdefined as the amount of enzyme capable of producing 1 μmol HMBAm at 20°C. for 1 minute in a reaction solution with a standard composition.

Specifically, for example, the enzymatic activity can be assayed by theprocedure as described in Examples. Further, protein quantification iscarried out by the dye-binding method using a protein assay kit fromBio-Rad.

On the other hand, as used herein, “cyanide resistance” means that theenzyme retains 40% or more nitrile hydratase activity when treated at20° C. for 30 minutes in the presence of 1 mM cyanide ion.Alternatively, when the enzyme retains 10% or more nitrile hydrataseactivity after treated at 20° C. for 30 minutes in the presence of 5 mMcyanide ion, one can state that the enzyme is cyanide-resistant.

Further, the present invention provides nitrile hydratase having thefollowing physicochemical properties (1)-(7):

(1) Molecular Weight:

The molecular weight is approximately 110,000 Da when determined by gelfiltration;

The enzyme is separated to two subunits of 26.8 kDa and 29.5 kDa bySDS-polyacrylamide gel electrophoresis.

(2) Action:

The enzyme acts on the nitrile group of nitrile compound.

(3) Optimal pH:

The activity of hydrating nitrile group is maximized at pH 5.5-6.5.

(4) Optimal Temperature:

The activity of hydrating nitrile group is maximized at 40-45° C.

(5) pH Stability:

The enzyme is stable within pH 4-9.

(6) Thermal Stability:

The enzyme retains 70% or more activity after heat-treated at 50° C. for30 minutes.

(7) Inhibitor:

The enzyme is inhibited by HgCl₂, AgNO₃, hydroxylamine orphenylhydrazine.

Further, the present invention provides nitrile hydratase having thefollowing physicochemical properties (c) and/or (d) in addition to theabove-mentioned physicochemical properties (a) and (b), or (1)-(7)

(c) Substrate Specificity:

The enzyme uses 2-hydroxy-4-methylthiobutyronitrile as the substrate andproduces 2-hydroxy-4-methylthiobutyroamide.

(d) Stabilization:

The enzyme is stabilized by divalent metal ions.

In the present invention, the substrate specificity of nitrile hydratasecan be determined by the method for testing the activity nitrilehydratase as described above. Further, as used herein, “stabilized bydivalent metal ions” means that the enzymatic activity is notsubstantially reduced even when the enzyme is incubated with 1.8 w/w %2-hydroxy-4-methylthbutyronitrile in the presence of a divalent metalion for 20 minutes. In a preferred embodiment of the present invention,the nitrile hydratase of the present invention can maintain 110% orhigher activity under these conditions.

In the present invention, the above-mentioned divalent metal ionincludes nickel ion and cobalt ion. These divalent metal ions enhancethe enzymatic activity of nitrile hydratase of the present invention ata concentration of 0.1 mM-1 M, generally 0.5-100 mM, preferably 1-10 mM.

The nitrile hydratase of the present invention can be purified frommicroorganisms capable of producing the enzyme by commonly used proteinpurification methods. The above microorganisms can be cultured in astandard bacterial culture medium. Some compounds for inducing theexpression of nitrile hydratase can be added to the medium. For example,the addition of a nitrile compound or amide compound can enhance theactivity of nitrile hydratase. More specifically, acetonitrile,acetamide and such can be used as the enzyme-inducer.

Microorganisms capable of producing the enzyme are sufficiently grown,and then the cells are harvested. The cells are lysed in an appropriatebuffer to prepare cell-free extract. The buffer can contain a reducingagent such as 2-mercaptoethanol, protease inhibitor such asphenylmethanesulfonyl fluoride (PMFS). The nitrile hydratase can bepurified from the cell-free extract by fractionation based on theprotein solubility and appropriate combinations of variouschromatographic procedures.

As a fractionation method based on the protein solubility, for example,precipitation with an organic solvent such as acetone anddimethylsulfoxide or salting out with ammonium sulfate can be used. Onthe other hand, known chromatographic methods include cation-exchangechromatography, anion-exchange chromatography, gel filtration,hydrophobic chromatography as well as many procedures of affinitychromatography using dye, antibody and others. More specifically, thenitrile hydratase of the present invention can be purified as anelectrophoretically homogeneous polypeptide, for example, by hydrophobicchromatography using phenyl-TOYOPEARL, anion-exchange chromatographyusing DEAE-Sepharose, hydrophobic chromatography using butyl-TOYOPEARL,affinity chromatography using Blue-Sepharose, gel filtration usingSuperdex 200, and others.

Microorganisms that can be used for this purpose include, for example,those belonging to the genus Rhodococcus (Rhodococcus sp.) Morespecifically, Rhodococcus sp. Cr4 is a suitable microorganism forproducing the nitrile hydratase of the present invention. Rhodococcussp. Cr4 has been deposited under the accession number of FERM BP-6596 inthe International Patent Organism Depositary.

International Deposition of Rhodococcus sp. Cr4:

-   (a) Name and Address of Depositary Institute    -   Name: International Patent Organism Depositary, National        Institute of Advanced Industrial Science and Technology (AIST),        Independent Administrative Institution (Previous Name: The        National Institute of Bioscience and Human-Technology, The        Agency of Industrial Science and Technology, The Ministry of        International Trade and Industry)    -   Address: Chuo 6, 1-1, Higashi 1-chome, Tsukuba-shi, Ibaraki        305-8566 Japan-   (b) Date of Deposition (Original Date of Deposition): Dec. 8, 1998-   (c) Accession Number: FERM BP-6596

The nitrile hydratase of the present invention is that can be obtainedfrom Rhodococcus sp. Cr4 is a novel enzyme having the above-mentionedphysicochemical properties (a)-(d) and (1)-(7). The structural featureis described above as the physicochemical property (1); the enzyme is ahetero-dimeric polypeptide consisting of α-subunit of 26.8 kDa andβ-subunit of 29.5 kDa, determined by SDS-PAGE. The amino acid sequenceof α-subunit is shown in SEQ ID NO: 2 (226 amino acid residues) and theamino acid sequence of β-subunit, in SEQ ID NO: 4 (207 amino acidresidues). Namely, the present invention provides the followingsubstantially pure protein complex having the nitrile hydrataseactivity.

The present invention relates to a substantially pure protein complexbetween polypeptide comprising the amino acid sequence of SEQ ID NO: 2and polypeptide comprising the amino acid sequence of SEQ ID NO: 4.Further the present invention includes a homologue of the proteincomplex between polypeptide comprising the amino acid sequence of SEQ IDNO: 2 and polypeptide comprising the amino acid sequence of SEQ ID NO:4.

The term “substantially pure” as used herein in reference to a givenprotein, polypeptide, or protein complex means that the protein,polypeptide, or protein complex is substantially free from otherbiological macromolecules. The substantially pure protein, polypeptide,or protein complex is at least 75% (e.g., at least 0.80, 85, 95, or 99%)pure by dry weight. Purity can be measured by any appropriate standardmethod known in the art, for example, by column chromatography,polyacrylamide gel electrophoresis, or HPLC analysis.

The protein complex of the present invention is a nitrile hydratase thatcan be expressed by genetic recombination techniques using isolatedpolynucleotides encoding the nitrile hydratase of the present invention.The polynucleotides encoding the nitrile hydratase of the presentinvention can be isolated, for example, by the following method.

The nitrile hydratase provided by the present invention consists ofα-subunit encoded by the polynucleotide shown in any one of (A)-(E), andβ-subunit encoded by the polynucleotide shown in any one of (a)-(e), andis a protein complex having the following physicochemical properties (i)and (ii).

(i) Effect:

Acting on the nitrile group of nitrile compound, hydrating the nitrilegroup and converting it to an amide group; and

(ii) Substrate Specificity:

The enzyme uses 2-hydroxy-4-methylthiobutyronitrile as the substrate andproduces 2-hydroxy-4-methylthiobutyroamide.

The polynucleotide shown in any one of the following (A)-(E) can be usedas the gene encoding α-subunit constituting the protein complex of thepresent invention. In the present invention, the polynucleotide shown inany one of (A)-(E) is useful for expressing the α-subunit of nitrilehydratase of the present invention:

-   -   (A) a polynucleotide comprising the nucleotide sequence of SEQ        ID NO: 1;    -   (B) a polynucleotide encoding polypeptide comprising the amino        acid sequence of SEQ ID NO: 2;    -   (C), a polynucleotide encoding polypeptide comprising the amino        acid sequence of SEQ ID NO: 2, which contains one or more amino        acid substitutions, deletions, insertions and/or additions;    -   (D) a polynucleotide capable of hybridizing to a polynucleotide        comprising the nucleotide sequence of SEQ ID NO:1 under        stringent conditions;    -   (E) a polynucleotide encoding polypeptide having 70% or higher        identity to the amino acid sequence of SEQ ID NO: 2.

Moreover, the polynucleotide shown in any one of the following (a)-(e)can be used as the gene encoding β-subunit constituting the proteincomplex of the present invention. In the present invention, thepolynucleotide shown in any one of (a)-(e) is useful for expressing theα-subunit of nitrile hydratase of the present invention:

(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 3;

(b) a polynucleotide encoding polypeptide comprising the amino acidsequence of SEQ ID NO: 4;

(c) a polynucleotide encoding polypeptide comprising the amino acidsequence of SEQ ID NO: 4, which contains one or more amino acidsubstitutions, deletions, insertions and/or additions;

(d) a polynucleotide capable of hybridizing to a polynucleotidecomprising the nucleotide sequence of SEQ ID NO: 3 under stringentconditions;

(e) a polynucleotide encoding polypeptide having 70% or higher identityto the amino acid sequence of SEQ ID NO: 4.

The present invention relates to isolated polynucleotides encodingsubunits of the nitrile hydratase and homologues thereof.

As used herein, an “isolated polynucleotide” is a polynucleotide thestructure of which is not identical to that of any naturally occurringpolynucleotide or to that of any fragment of a naturally occurringgenomic polynucleotide spanning more than three separate genes. The termtherefore includes, for example, (a) a DNA which has the sequence ofpart of a naturally occurring genomic DNA molecule in the genome of theorganism in which it naturally occurs; (b) a polynucleotide incorporatedinto a vector or into the genomic DNA of a prokaryote or eukaryote in amanner such that the resulting molecule is not identical to anynaturally occurring vector or genomic DNA; (c) a separate molecule suchas a cDNA, a genomic fragment, a fragment produced by polymerase chainreaction (PCR), or a restriction fragment; and (d) a recombinantnucleotide sequence that is part of a hybrid gene, i.e., a gene encodinga fusion polypeptide. Specifically excluded from this definition arepolynucleotides of DNA molecules present in mixtures of different (i)DNA molecules, (ii) transfected cells, or (iii) cell clones; e.g., asthese occur in a DNA library such as a cDNA or genomic DNA library.

Accordingly, in one aspect, the invention provides an isolatedpolynucleotide that encodes a polypeptide described herein or a fragmentthereof. Preferably, the isolated polynucleotide includes a nucleotidesequence that is at least 60% identical to the nucleotide sequence shownin SEQ ID NO: 1 or 3. More preferably, the isolated nucleic acidmolecule is at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 0.93%, 94%,0.95%, 96%, 97%, 98%, 99%, or more, identical to the nucleotide sequenceshown in SEQ ID NO: 1 or 3. In the case of an isolated polynucleotidewhich is longer than or equivalent in length to the reference sequence,e.g., SEQ ID NO: 1 or 3, the comparison is made with the full length ofthe reference sequence. Where the isolated polynucleotide is shorterthan the reference sequence, e.g., shorter than SEQ ID NO: 1 or 3, thecomparison is made to segment of the reference sequence of the samelength (excluding any loop required by the homology calculation).

Each of the nucleotide sequences of polynucleotides encoding α-subunitand β-subunit of nitrile hydratase of the present invention as well asthe amino acid sequences encoded by the nucleotide sequences are novel.Based on the information of the nucleotide sequences revealed in thepresent invention, the genes of interest can be obtained form thedeposited microorganism as described above. The genes can be obtained byPCR or screening with hybridization technique. The full-length genes canalso be obtained by chemical DNA synthesis.

Further, based on the information on the above nucleotide sequences, itis possible to obtain the nitrile hydratase gene derived from otherorganisms. For example, the nitrile hydratase genes derived from variousorganisms can be isolated by performing, under stringent conditions,hybridization to polynucleotides prepared from other organisms, usingthe above nucleotide sequence or a partial sequence thereof as a probe.

The term “polynucleotide capable of hybridizing under stringentconditions” means a polynucleotide capable of hybridizing to apolynucleotide, which has a nucleotide sequence selected from thenucleotide sequences of SEQ ID NO: 1 (α-subunit) and SEQ ID NO: 3(β-subunit), as a probe, for example, with an ECL direct nucleic acidlabeling and detection system (Amersham Pharmacia Biotech) under theconditions as described in the manual (wash: with a primary wash buffercontaining 0.5×SSC at 42° C.). Also included in the invention is apolynucleotide that hybridizes under high stringency conditions to thenucleotide sequence of SEQ ID NO: 1 or a segment thereof as describedherein. “High stringency conditions” refers to hybridization in 6×SSC atabout 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65°C. The nucleotide sequence constituting the probe polynucleotide can beprepared as one or more sequences consisting of arbitrary but at leastconsecutive 20 nucleotides, preferably at least 30 consecutivenucleotides, for example, consecutive 40, 60 or 100 nucleotides selectedfrom the above-mentioned nucleotide sequences.

Further, based on the information on the above nucleotide sequences, PCRprimers can be designed from regions exhibiting high homology. The geneencoding nitrile hydratases can be isolated from various organisms byPCR using such primers and chromosomal DNA or cDNA as a template.

In the method of the present invention, it is possible to use not onlythe natural enzyme but also an enzyme comprising the amino acid sequenceof the natural enzyme in which one or more amino acids have beensubstituted, deleted, and/or inserted, when the enzyme can form theprotein complex having the above-mentioned physicochemical properties(a) and (b) or (1) to (7). One skilled in the art can modify thestructure of the polypeptide, for example, via introducing mutations ofappropriate substitutions, deletions, insertions and/or additions bysite-specific mutagenesis (Nucleic Acid Res. 10, pp. 6487 (1982);Methods in Enzymol. 100, pp. 448 (1983); Molecular Cloning 2nd Edt.,Cold Spring Harbor Laboratory Press (1989.); PCR, A Practical ApproachIRL Press pp. 200 (1991)) and such. Further, amino acid mutations can begenerated in nature. Thus, not only the enzyme having artificial aminoacid mutations but also the enzyme containing spontaneous amino acidmutations can be used in the method of the present invention.

The number of amino acids that are mutated is not particularlyrestricted, as long as the enzyme can form the protein complex havingthe above-mentioned physicochemical properties (a) and (b) or (1) to(7). Normally, it is within 50 amino acids, preferably within 30 aminoacids, more preferably within 10 amino acids, and even more preferablywithin 3 amino acids. The site of mutation may be any site, as long asthe enzyme can form the protein complex having the above-mentionedphysicochemical properties (a) and (b) or (1) to (7).

An amino acid substitution is preferably mutated into different aminoacid(s) in which the properties of the amino acid side-chain areconserved. A “conservative amino acid substitution” is a replacement ofone amino acid residue belonging to one of the following groups having achemically similar side chain with another amino acid in the same group.Groups of amino acid residues having similar side chains have beendefined in the art. These groups include amino acids with basic sidechains (e.g., lysine, arginine, histidine), acidic side chains (e.g.,aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine).

In addition in the method of the present invention, the gene encoding apolypeptide having homology to the amino acid sequence of each subunitof nitrile hydratase can also be used for the present invention, whenthe product can form the protein complex having the above-mentionedphysicochemical properties (a) and (b) or (1)-(7). The genes can beobtained by using protein homology search. Such homology search can becarried out, for example, by using the following publicly knowndatabases:

Amino acid sequence databases for protein such as SWISS-PROT and PIR,

DNA databases such as DNA Databank of JAPAN (DDBJ), EMBL and GenBank,

Databases of amino acid sequences deduced from DNA sequences, and,

Programs for homology search such as FASTA program and BLAST program.

Further, database search services using the programs for searching theabove databases are also available on Internet. The nitrile hydratase tobe used in the present invention can be found by using this type ofservice.

A polypeptide having at least 85%, preferably 90% or higher, morepreferably 95% or higher identity to the amino acid sequences of SEQ IDNO: 2 (α-subunit) or SEQ ID NO: 4 (β-subunit) is a preferred polypeptideof the present invention constituting the nitrile hydratase to be usedin the present invention. As used herein, “percent identity” means, forexample, the value of percent identity in “Positive” using BLASTprogram. Specifically, “percent identity” of two amino acid sequences orof two nucleic acids is determined using the algorithm of Karlin andAltschul (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990) modified as inKarlin and Altschul (Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993).Such an algorithm is incorporated into the NBLAST and XBLAST programs ofAltschul et al. (J. Mol. Biol. 215:403-410, 1990). BLAST nucleotidesearches are performed with the NBLAST program, score=100,wordlength=12. Homology search of protein can readily be performed, forexample, in DNA Databank of JAPAN (DDBJ), by using the FASTA program,BLAST program, etc. BLAST protein searches are performed with the XBLASTprogram, score=50, wordlength=3. Where gaps exist between two sequences,Gapped BLAST is utilized as described in Altsuchl et al. (Nucleic AcidsRes. 25: 3389-3402, 1997). When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g, XBLASTand NBLAST) are used.

In the present invention, when the protein complex is intended to beprepared by using mutant α-subunit comprising the amino acid sequence ofSEQ ID NO: 2 and mutant β-subunit comprising the amino acid sequence ofSEQ ID NO: 4 both of which have mutations in their amino acid sequences,either or both of them may be mutant polypeptides. Further, the subunitsof different origins can be combined with each other to form the proteincomplex of the present invention. When one of the two subunits isintended to be a mutant, it should be selected to be capable ofconstituting the protein complex, which has the above-mentionedphysicochemical properties (1) and (2), with the other subunit.

Further, when both α-subunit and β-subunit are intended to be mutants,one mutant is combined with the other, and mutants capable ofconstituting the protein complex that has the above-mentionedphysicochemical properties (i) and (ii) are selected as well. In thiscase, first, a mutant subunit is tested to assess whether it gives therequired physicochemical properties in combination with the othersubunit having the wild-type amino acid sequence when the result ispositive, another mutant, which gives the required physicochemicalproperties, is selected for the partner subunit that is used incombination with the first mutant; thus the mutants are readilyselectable. The desirable mutants of the present invention have theabove-mentioned physicochemical properties (a) and (b) or (1)-(7) aswell as the above-mentioned physicochemical properties (i) and (ii).

Preferable enzymatically active material of the present inventionincludes a transformant of homologous or heterogonous host expressingthe gene encoding nitrile hydratase, which has been prepared by geneticrecombination techniques and treated products thereof.

There is no limitation on the organism to be used for the transformationto express the nitrile hydratase gene of the present invention, when theorganism can be transformed with the recombinant vector containingpolynucleotides encoding the respective subunits constituting theprotein complex having the nitrile hydratase activity and can expressthe nitrile hydratase activity. The polynucleotides encoding therespective subunits can be retaining in a single vector. Thepolynucleotides for the respective subunits can also inserted separatelyin two types of vectors; the protein complex of the present inventioncan be expressed by the co-transformation of the vectors. Further, it ispossible to obtain the protein complex of interest in vitro by combiningthe transformants each of which expresses a single subunit of the two.Available microorganisms are those for which host-vector systems areavailable and include, for example:

bacteria such as the genus Escherichia, the genus Bacillus, the genusPseudomonas, the genus Serratia, the genus Brevibacterium, the genusCorynebacteriun, the genus Streptococcus, and the genus Lactobacillus;

actinomycetes such as the genus Rhodococcus and the genus Streptomyces;

yeasts such as the genus Saccharomyces, the genus Kluyveromyces, thegenus Schizosaccharomyces, the genus Zygosaccharomyces, the genusYarrowia, the genus Trichosporon, the genus Rhodosporidium, the genusPichia, and the genus Candida; and,

fungi such as the genus Neurospora, the genus Aspergillus, the genusCephalosporium, and the genus Trichoderma, etc.

Procedure for preparation of a transformant and construction of arecombinant vector suitable for a host can be carried out by employingtechniques that are commonly used in the fields of molecular biology,bioengineering, and genetic engineering (for example, see Sambrook etal., “Molecular Cloning”, Cold Spring Harbor Laboratories). In order toexpress the gene encoding nitrile hydratase of the present invention ina microorganism, it is necessary to introduce the polynucleotide into aplasmid vector or phage vector that is stable in the microorganism andto let the genetic information transcribed and translated. To do so, apromoter, a unit for regulating transcription and translation, is placedupstream of the 5′-end of the polynucleotide of the present invention,and preferably a terminator is placed downstream of the 3′-end of thepolynucleotide. The promoter and the terminator should be functional inthe microorganism to be utilized as a host. Available vectors,promoters, and terminators for the above-mentioned variousmicroorganisms are described in detail in “Fundamental Course inMicrobiology (8): Genetic Engineering”, Kyoritsu Shuppan, specificallyfor yeasts, in “Adv. Biochem. Eng. 43, 75-102(1990)” and “Yeast 8,423-488 (1992).”

For example, for the genus Escherichia, in particular, for Escherichiacoli, available plasmids include pBR series and pUC series plasmids;available promoters include promoters derived from lac (derived fromβ-galactosidase gene), trp (derived from the tryptophan operon), tac andtrc (which are chimeras of lac and trp), PL and PR of λ phage, etc.Available terminators are derived from trpA, phages, rrnB ribosomal RNA,etc. Among these, a vector pSE420D (described in Unexamined PublishedJapanese Patent Application No. (JP-A) 2000-189170), which isconstructed by partially modifying the multicloning site of commerciallyavailable pSE420 (Invitrogen), can be preferably used.

For the genus Bacillus, available vectors are pUB110 series and pC194series plasmids; the vectors can be integrated into host chromosome.Available promoters and terminators are derived from apr (alkalineprotease), npr (neutral protease), amy (α-amylase), etc.

For the genus Pseudomonas, there are host-vector systems developed forPseudomonas putida and Pseudomonas cepacia. A broad-host-range vector,pKT240, (containing RSF1010-derived genes required for autonomousreplication) based on TOL plasmid, which is involved in decomposition oftoluene compounds, is available; a promoter and a terminator derivedfrom the lipase gene (JP-A Hei 5-284973) are available.

For the genus Brevibacterium, in particular, for Brevibacteriumlactofermentum, available plasmid vectors include pAJ43 (Gene 39, 281(1985)). Promoters and terminators used for Escherichia coli can beutilized without any modification for Brevibacterium.

For the genus Corynebacterium, in particular, for Corynebacteriumglutamicum, plasmid vectors such as pCS11 (JP-A Sho 57-183799) andpCB101 (Mol. Gen. Genet. 196, 175(1984)) are available.

For the genus Streptococcus, plasmid vectors such as pHV1301 (FEMSMicrobiol. Lett. 26, 239 (1985)) and pGK1 (Appl. Environ. Microbiol. 50,94 (1985)) can be used.

For the genus Lactobacillus, plasmid vectors such as pAMβ1 (J.Bacteriol. 137, 614 (1979)), which was developed for the genusStreptococcus, can be utilized; and promoters that are used forEscherichia coli are also usable.

For the genus Rhodococcus, plasmid vectors isolated from Rhodococcusrhodochrous are available (J. Gen. Microbiol. 138, 1003 (1992)).

For the genus Streptomyces, plasmids can be constructed in accordancewith the method as described in “Genetic Manipulation of Streptomyces: ALaboratory Manual” (Cold Spring Harbor Laboratories (1985)) by Hopwoodet al. In particular, for Streptomyces lividans, pIJ486 (Mol. Gen.Genet. 203, 468-478, 1986), pKC1064 (Gene 103,97-99 (1991)), and pUWL-KS(Gene 165, 149-150 (1995)) are usable. The same plasmids can also beutilized for Streptomyces virginiae (Actinomycetol. 11, 46-53 (1997)).

For the genus Saccharomyces, in particular, for Saccharomycescerevisiae, YRp series, YEp series, YCp series, and YIp series plasmidsare available; integration vectors (refer EP 537456, etc.) which areintegrated into chromosome via homologous recombination withmulticopy-ribosomal genes, allow to introduce a gene of interest inmulticopy and the gene incorporated is stably maintained in themicroorganism; and thus, these types of vectors are highly useful.Available promoters and terminators are derived from genes encoding ADH(alcohol dehydrogenase), GAPDH (glyceraldehyde-3-phosphatedehydrogenase), PHO (acid phosphatase), GAL (β-galactosidase), PGK(phosphoglycerate kinase), ENO (enolase), etc.

For the genus Kluyveromyces, in particular, for Kluyveromyces lactis,available plasmids are those such as 2-μm plasmids derived fromSaccharomyces cerevisiae, pKD1 series plasmids (J. Bacteriol. 145,382-390(1981)), plasmids derived from pGKl1 and involved in the killeractivity, KARS (Kluyveromyces autonomous replication sequence) plasmids,and plasmids (refer EP 537456, etc.) capable of being integrated intochromosome via homologous recombination with the ribosomal DNA.Promoters and terminators derived from ADH, PGK, and such are available.

For the genus Schizosaccharomyces, it is possible to use plasmid vectorscomprising ARS (autonomous replication sequence) derived fromSchizosaccharomyces pombe and auxotrophy-complementing selectablemarkers derived from Saccharomyces cerevisiae (Mol. Cell. Biol. 6, 80(1986)). Promoters such as ADH promoter derived from Schizosaccharomycespombe are usable (EMBO J. 6, 729 (1987)) In particular, pAUR224 iscommercially available from TaKaRa Shuzo.

For the genus Zygosaccharomyces, plasmids originating from those such aspSB3 (Nucleic Acids Res. 13, 4267 (1985)) derived from Zygosaccharomycesrouxii are available; it is possible to use promoters such as PHO5promoter derived from Saccharomyces cerevisiae and GAP-Zr(Glyceraldehyde-3-phosphate dehydrogenase) promoter (Agri. Biol. Chem.54, 2521 (1990)) derived from Zygosaccharomyces rouxii.

For the genus Pichia, host vector system where Pichia-derived genesinvolved in autonomous replication (PARS1 and PARS2) are used in Pichiapastoris and such has been developed (Mol. Cell. Biol. 5, 3376 (1985)),and thus high-density cultivation and strong promoters such asmethanol-inducible AOX are usable (Nucleic Acids Res. 15, 3859 (1987)).A Host-vector system has been developed for Pichia angusta (previouslycalled Hansenula polyorpha) among the genus Pichia. Usable vectorsinclude Pichia angusta-derived genes (HARS1 and HARS2) involved inautonomous replication, but they are relatively unstable. Therefore,multi-copy integration of the gene into a chromosome is effective (Yeast7, 431-443 (1991)). Promoters of AOX (alcohol oxidase) and FDH. (formicacid dehydrogenase), which are induced by methanol and such, are alsoavailable. Another host vector system where Pichia-derived genesinvolved in autonomous replication (PARS1 and PARS2) are used in Pichiapastoris and such has been developed (Mol. Cell. Biol. 5, 3376 (1985)),and thus high-density cultivation and strong promoters such asmethanol-inducible AOX are usable (Nucleic Acids Res. 15, 3859 (1987)).

For the genus Candida, host-vector systems have been developed forCandida maltosa, Candida albicans, Candida tropicalis, Candida utilis,etc. An autonomous replication sequence originating from Candida maltosahas been cloned (Agri. Biol. Chem. 51, 51,1587 (1987)), and a vectorusing the sequence has been developed for Candida maltosa. Further, achromosome-integration vector with a highly efficient promoter unit hasbeen developed for Candida utilis (JP-A Hei. 08-173170).

For the genus Aspergillus, Aspergillus niger and Aspergillus oryzae haveintensively been studied among fungi, and thus plasmid vectors andchromosome-integration vectors are available, as well as promotersderived from an extracellular protease gene and amylase gene (Trends inBiotechnology 7, 283-287 (1989)).

For the genus Trichoderma, host-vector systems have been developed forTrichoderma reesei, and promoters such as that derived from anextracellular cellulase gene are available. (Biotechnology 7,596-603(1989)).

There are various host-vector systems developed for plants and animalsother than microorganisms; in particular, the systems include those ofinsect such as silkworm (Nature 315, 592-594(1985)), and plants such asrapeseed, maize, potato and such are preferably usable. The culture oftransformant and purification of nitrile hydratase from the transformantcan be carried out by methods known to one skilled in the art.

Polynucleotides encoding nitrile hydratase to be used as inserts in thevector of the present invention include, for example, any one of theabove polynucleotides shown in (A)-(E) and any one of the abovepolynucleotides shown in (a)-(e).

The polynucleotides encoding α-subunit and β-subunit of nitrilehydratase in the vector of the present invention are preferably linkedin tandem. The term. “preferably linked in tandem” means the linkagesuch that a common regulatory region directs the expression of thesesubunits. Such an arrangement is expected to enable more efficientexpression of the subunits and more efficient formation of the proteincomplex having the enzymatic activity. Alternatively, thepolynucleotides for the respective subunits can also be insertedseparately in two types of vectors; the protein complex can be expressedby the co-transformation of the two vectors.

The present invention also relates to transformants of the presentinvention retaining the vector in an expressible manner. The vector ofthe present invention can be transformed into an arbitrary host, whenthe host can retain the vector in a functional form. Such a host that isusable for this purpose includes, for example, E. coli.

The nitrile hydratase of the present invention, microorganism producingthe nitrile hydratase, protein complex of the present invention,transformant producing the protein complex and treated products thereofare useful for the method to produce amides using nitrile compounds asthe substrates. Namely, the present invention provides a method forproducing amides, the method comprising the step of recovering theamides by contacting nitrile compounds with the enzymatically activematerial selected from the group consisting of nitrile hydratase of thepresent invention, microorganism producing the nitrile hydratase,protein complex of the present invention, transformant producing theprotein complex and processed products thereof.

As used herein, the term “nitrile hydratase” means an enzyme having theabove-mentioned physicochemical properties (a) and (b) or enzyme havingthe above-mentioned physicochemical properties (1)-(7). Further, themicroorganism capable of producing the enzyme includes the strainRhodococcus sp. Cr4 from which the enzyme is derived, microorganismsbelonging to the genus Rhodococcus producing the enzyme of the presentinvention and transformed host microorganism containing thepolynucleotide encoding the enzyme. Further, the term “transformed hostmicroorganism” means a host microorganism capable of expressing thepolynucleotides (A)-(E) encoding the above-mentioned α-subunit and/orpolynucleotides (a)-(e) encoding β-subunit. The above-mentioned hostmicroorganism can produce the protein complex of the present inventionconsisting of α-subunit and β-subunit of the present invention.

Further, the treated product of microorganism specifically includesmicroorganism of which cell membrane permeability has been modified bythe treatment with a detergent or organic solvent such as toluene,cell-free extract obtained by lysing the cells by the treatment withglass beads or enzyme and material partially purified from the extract,etc. Alternatively, the treated product of the enzyme includes theenzyme linked with insoluble carrier or with aqueous carrier moleculeand the immobilized enzyme molecules prepared by immobilize entrapping,etc.

In the present invention, the enzymatically active material includes allmaterials having the enzymatic activity of nitrile hydratase of thepresent invention. Accordingly, as long as a material has the desiredenzymatic activity, it is included in the enzymatically active material,regardless of its enzymatic purity and solubility.

The enzyme reaction constituting the amide-producing method of thepresent invention can be carried out by contacting the above-mentionedenzymatically active material with a reaction solution containing anitrile compound as the substrate. Specifically, the enzymaticallyactive material can be contacted with the substrate in an aqueoussolvent, mixed solvent consisting of aqueous solvent and water-solubleorganic solvent, or two-phase system with a water-insoluble solvent. Theaqueous solvent includes buffers having the buffering action at neutralpH such as phosphate buffer and Tris-HCl buffer. Alternatively, when thepH changes can be within a desirable range during the reaction by usingan acid and alkali, no buffer is needed. The organic solvent immisciblewith water includes, for example, ethyl acetate, butyl acetate, toluene,chloroform, n-hexane and isooctane, etc. The reaction can be conductedin a mixed solvent, which consists of an aqueous solvent, and organicsolvent such as ethanol, acetone, dimethylsulfoxide and acetonitrile.

In the two-phase system, the enzymatically active material is present inthe aqueous phase in which the material is used without any othersolvent or combined with water or a buffer. The substrate compound canbe dissolved in an aqueous solvent such as water, buffer and ethanol,and supplied to the reaction system. In this case, along with theenzymatically active material, the substrate constitutes a single-phasereaction system. In addition, the reaction of the present invention canbe carried out by using immobilized enzyme, membrane reactor, etc.Furthermore, the transformant of the present invention can be used inthe form of the culture, cells separated from the culture medium byfiltration, centrifugation or the like, or cells resuspended in buffer,water, or the like after they are separated by centrifugation andwashed. The separated cells can be used in a state as they arerecovered, as their disrupts, as treated with acetone or toluene, or aslyophilizate. When the enzyme is extracellularly produced, the culturemedium of the transformant can also be used after it is separated fromthe transformant by the usual methods. Forms of contacting theenzymatically active material with the reaction solution are not limitedto these Examples. The reaction solution means a solution consisting ofthe substrate dissolved in an appropriate solvent that provides suitableenvironment for the expression of enzymatic activity.

There is no limitation on the type of nitrile compound to be used in themethod for producing amides using the nitrile hydratase of the presentinvention. For example, the following nitrile compounds can be used inthe method of the present invention.

Saturated mononitriles;

acetonitrile, propionitrile, butyronitrile, isobutyronitrile,valeronitrile, isovaleronitrile, capronitrile, etc.

Saturated dinitriles;

malonitrile, succinonitrile, glutarnitrile, adiponitrile, etc.

α-aminonitriles;

α-aminopropionitrile, α-aminomethylthiobutyronitrile,α-aminobutyronitrile, aminoacetonitrile, etc.

Nitriles having carboxyl groups;

cyanoacetic acid, etc.

β-aminonitriles;

amino-3-propionitrile, etc.

Unsaturated nitrites;

acrylonitrile, methacrylonitrile, cyanoallyl, crotonitrile, etc.

Aromatic nitrites;

benzonitrile, o-, m- and p-chlorobenzonitrile, o-, m- andp-fluorobenzonitrile, o-, m- and p-nitrobenzonitrile,p-aminobenzonitrile, 4-cyanophenol, o-, m- and p-tolunitrile,2,4-dichlorobenzonitrile, 2,6-dichlorobenzonitrile,2,6-difluorobenzonitrile, anisonitrile, α-naphthonitrile,β-naphthonitrile, phthalonitrile, isophthalonitrile, terephthalonitrile,cyanobenzyl, phenylacetonitrile, etc.

α-hydroxynitriles.

In the present invention, particularly preferable nitrile compoundsinclude α-hydroxynitrile compound. In the amide-producing method of thepresent invention, there is no limitation on the type ofα-hydroxynitrile compound. More specifically, for example, the compoundrepresented by the above formula (1) can be used.

In the formula, R represents, substituted or unsubstituted alkyl group,substituted or unsubstituted alkenyl group, substituted or unsubstitutedcycloalkyl group, substituted or unsubstituted alkoxy group, substitutedor unsubstituted aryl group, substituted or unsubstituted aryloxy group,substituted or unsubstituted saturated or unsaturated heterocyclicgroup. α-hydroxyamide can be produced from the α-hydroxynitrilecompounds.

The heterocyclic group includes the groups having at least one ofnitrogen, oxygen and sulfur as the heteroatom. Further, the substituentincludes, for example, alkyl group, alkoxy group acyl group, aryl group,aryloxy group, halogens such as chloride and bromide, hydroxy group,amino group, nitro group, thiol group, etc.

Specifically, for example, the following compounds or substitutedproducts thereof can be used. As used herein, the substituted productmeans compounds having the substituents as exemplified above.

Lactonitrile

α-hydroxy-n-propionitrile

α-hydroxy-n-butyronitrile

α-hydroxy-isobutyronitrile

α-hydroxy-n-hexyronitrile

α-hydroxy-n-heptyronitrile

α-hydroxy-n-octyronitrile

α,γ-dihydroxy-β,β-dimethylbutyronitrile

Acroleincyanohydrin

Methacrylaldehyde cyanohydrin

3-chlorolactonitrile

4-methylthio-α-hydroxybutyronitrile

α-hydroxy-α-phenylpropionyl

In addition, the substrate compound having aromatic ring and heterocycleinclude the following exemplary compounds and substituted productsthereof.

Mandelonitrile

2-thiophenecarboxyaldehyde cyanohydrin

2-pyridinecarboxyaldehyde cyanohydrin

2-pyrrolecarboxyaldehyde cyanohydrin

2-furaldehyde cyanohydrin

2-naphthylaldehyde cyanohydrin

Many of the nitrile compounds represented by the α-hydroxynitrilecompound of formula (1) are decomposed into aldehyde and hydrocyanicacid in a polar solvent. For example, the α-hydroxynitrile compound offormula (1) is converted to the aldehyde and hydrocyanic acid of thefollowing formula (2).R—CHO  (2)Since a state of equilibrium is established among these compounds, theconsumption of α-hydroxynitrile compound by enzyme reaction shifts theequilibrium toward the α-hydroxynitrile compound.

On the other hand, cyanide and aldehyde derived from hydrocyanic acidmay give some damage to the enzyme polypeptide. Accordingly, previouslyknown nitrile hydratases cannot hydrate sufficient amounts ofα-hydroxynitrile compound due to their decreased enzymatic activity, andthus do not provide enough yields of the products. However, the nitrilehydratase of the present invention retains the enzymatic activity evenin the presence of cyanide or aldehyde. Thus, the enzyme can utilizenitrile compounds generated from aldehydes and hydrocyanic acids as thesubstrate. Accordingly, by the inventive method for producingα-hydroxyamide, the compound of formula (1) can be supplied from thealdehyde compound and hydrocyanic acid represented by the followingformula (2).R—CHO  (2)

The amide-producing method of the present invention is preferablyconducted in the presence of a divalent metal ion. The divalent metalion contributes to the activity of nitrile hydratase of the presentinvention. The preferable divalent metal ions include nickel ion andcobalt ion. These ions can be added to the reaction solution as anappropriate aqueous salt. Specifically, the ion can be added as achloride salt.

In the present invention, the hydration or hydrolysis of nitrilecompound can be achieved by contacting the enzymatically active materialof the present invention with a substrate compound, or a mixture ofaldehyde and hydrocyanic acid represented by formula (2), which can beconverted to a substrate compound in an aqueous solvent such as water orbuffer. The reaction solution preferably contains divalent metal ions.

As used herein, the term “hydration” means the reaction where watermolecules attaches to the nitrile group. Contrasted with “hydration”,the term-“hydrolysis” means the reaction where, from a compound in whicha substituent is linked with the nitrile group, the substituent iscleaved off by hydrolysis. Both reactions are included by theamide-producing method of the present invention.

There is no limitation on the concentration of substrate compound in thereaction solution. In order to prevent the inhibition of enzymaticactivity by the substrate compound, the concentration can correspond to,for example, 0.1-10 w/w % in general, preferably 0.2-5.0 w/w %, in thecase of α-hydroxynitrile. The substrate can be added once at the startof reaction, but it is preferable to add the substrate continuously ordiscontinuously to prevent the substrate concentration from being toohigh.

When the solubility of nitrile compound as the substrate in the aqueoussolvent is too low, a detergent can be added to the reaction solution.0.1-5.0 w/w % Triton X-100 or Tween 60 can be use as the detergent. Toincrease the substrate solubility, a mixed solvent containing an organicsolvent can be used effectively. Specifically, for example, the reactionefficiency can be improved by adding methanol, ethanol,dimethylsulfoxide, and such. Alternatively, the reaction of the presentinvention can be achieved in an organic solvent insoluble with water ortwo-phase system consisting of aqueous solvent and organic solventinsoluble with water. The organic solvent immiscible with water that isusable includes, for example, ethyl acetate, butyl acetate, toluene,chloroform, n-hexane, cyclohexane, octane or 1-octanol, etc.

When the substrate concentration falls within the range as describedabove, the efficient enzymatic reaction can be achieved by using nitrilehydratase of the present invention at an enzyme concentration, forexample, of 1 mU/mL-100 U/mL, preferably 100 MU/mL or higher. Further,when microorganism cells are used as the enzymatically active material,the amount of microorganism to be used relative to that of the substratepreferably ranges from 0.01 to 5.0 w/w % as dry cells. The enzymaticallyactive material such as an enzyme and cells can be contacted with thesubstrate by dissolving or dispersing them in a reaction solution.Alternatively, it is possible to use the enzymatically active materialimmobilized by the techniques of chemical linking or entrapment.Further, the reaction can be carried out in a state where the substratesolution is isolated from the enzymatically active material, with aporous membrane, through which the substrate is permeable, but whichlimits the permeation of the enzyme molecule or the cells.

The reaction can be carried out typically at a temperature ranging fromthe freezing point to 50° C., preferably at 10-30° C., for 0.1-100hours. There is no limitation on the pH of reaction solution, when theenzymatic activity can be maintained. Since the optimal pH of nitrilehydratase of the present invention ranges from 5.5 to 6.5, it ispreferable to adjust the pH of reaction solution within this range.

Thus, the nitrile compound is converted to a corresponding amide by thehydration or hydrolysis action of the microorganism, and accumulates inthe reaction solution. The produced amide can be recovered and purifiedfrom the reaction solution by appropriate methods. Specifically, forexample, the amide can be recovered and purified by the combined use oftypical methods such as ultrafiltration, concentration, columnchromatography, extraction, treatment with activated charcoal,distillation, etc.

The present invention provides a method for stabilizing the activity ofnitrile hydratase in the presence of the nitrile compound, where themethod is characterized by coexistence of divalent metal ions. Further,the present invention provides a method for producing amide, the methodcomprising the step of reacting the nitrile hydratase to the nitrilecompound in the presence of divalent metal ion and recovering the amidegenerated.

The present invention is based on the finding that the presence ofdivalent metal ion in the reaction system markedly suppresses thedecrease in the enzymatic activity of nitrile hydratase activity. In thepresent invention, there is no limitation on the origin of nitrilehydratase. Namely, any enzyme can be used, when the enzyme has theactivity of acting on and hydrating the nitrile group of nitrilecompound, and converting the nitrile group to the amide group. Thenitrile hydratase, which hydrates α-hydroxynitrile to the correspondingamide, is the preferred enzyme in the present invention.

It can be assumed that the divalent metal ion of the present inventionachieves the effect of suppressing the decrease in the nitrile hydrataseactivity, for example, by the following mechanism: At first, the bindingof metal ion to the cyanohydrin structure of substrate compound mayenhance the enzyme reaction. Secondly, the binding of metal ion mayprevent the dissociation of cyanohydrin. As a result, the concentrationof cyanide ion is reduced in the reaction solution. As the reducedcyanide ion concentration lowers the inhibitory effect on the enzyme,the enzymatic activity is raised.

In any case, it is assumed that these phenomena are not specific to someparticular nitrile hydratases but generally found in the uses ofsubstrate compounds having the cyanohydrin structure. This is the reasonwhy there is no limitation on the origin of nitrile hydratase to be usedin the method for stabilizing the activity of nitrile hydratase of thepresent invention or method for producing amides.

Known enzymes capable of hydrating nitrile compounds to thecorresponding amides include, for example, the enzymes derived form thefollowing microorganism (see JP-A Hei 04-040899).

The genus Rhodococcus

The genus Corynebacterium

The genus Pseudomonas

The genus Arthrobacter

The genus Alcaligenes

The genus Bacillus

The genus Bacteridium

The genus Micrococcus

The genus Brevibacterium

The genus Nocardia.

More specifically, for example, microorganisms include the following:

Rhodococcus rhodochrous ATCC 33278

Rhodococcus erythropolis IFO 12320

Corynebacterium nitrilophilus ATCC 21419

Pseudomonas sp. SK87 (FERM P-11311)

Arthrobacter sp. HR1 (FERM BP-3323)

Alcaligenes sp. BC16-2 (FERM BP-3321)

Rhodococcus sp. HT40-6 (FERM P-11774)

Microorganisms described in JP-B Sho 62-21519.

Other microorganisms are publicly known and available from American TypeCulture Collection (ATCC); Institute of Applied Microbiology (IAM), TheUniversity of Tokyo; Fermentation Research Institute, the Agency ofIndustrial Science and Technology; KAKEN PHARMACEUTICAL Co. (KCC);Institute for Fermentation, Osaka (IFO); and RESEARCH CENTER FORPATHOGENIC FUNGI AND MICROBIAL TOXICOSES (IFM), The University of Chiba.In addition, the nitrile hydratase of the present invention that can beobtained from the above-mentioned Rhodococcus sp. Cr4 (FERM BP-6596) isalso one of the preferred enzyme of the present invention.

There is no limitation on the nitrile compound to be used in theamide-producing method of the present invention. More specifically, thepreferred substrates include, for example, compounds illustrated in theabove description for the method for producing α-hydroxyamide with thenitrile hydratase of the present invention.

It is also possible to conveniently produce only either of the twooptically active isomers of α-hydroxyamide or α-hydroxy acid, the enzymecapable of stereospecifically hydrating or hydrolyzing nitrites or amicroorganism containing the enzyme is used in the reaction. Thus, bythe method of the present invention, the stereospecific α-hydroxyamideor α-hydroxy acid can be obtained much more advantageously than by theprevious methods producing it through the step of optical resolution orracemization.

In the present invention, the hydration or hydrolysis of nitrilecompound is carried out, for example, by contacting the nitrilehydratase with the α-hydroxynitrile represented by formula (1) in anaqueous solvent such as water or buffer. When the nitrile hydratase tobe used is resistant to cyanide and aldehyde, the reaction can also becarried out in the presence of the aldehyde and hydrocyanic acidrepresented by formula (2), which can be converted to α-hydroxynitrile.The nitrile hydratase may be a lysate of microorganism capable ofproducing the enzyme, crude enzyme, or products obtained by immobilizingthe materials, in addition to the purified enzyme. Further, in thepresent invention, the divalent metal ion may be added in the reactionsolution at a concentration of 0.1 mM-1 M, generally 0.5-100 mM,preferably 1-10 mM.

There is no limitation on the type of metal ion to be used in thepresent invention, when the ion can be effective for maintaining theactivity of nitrile hydratase. For example, cobalt ion and nickel ionare preferred metal ions, which can keep the activity of nitrilehydratase high. These metal ions can be added as salts such as chloridesalts in the reaction solution.

In the present invention, the reaction conditions can be adjustedappropriately depending on the properties of substrate compounds to beused in combination with the nitrile hydratase, in addition to thepresence of divalent metal ion. There is no limitation on the type ofsubstrate compound and reaction conditions to be used in the reaction.Specifically, for example, the reaction system can be adjusted based onthe exemplary conditions illustrated for the method for producing theα-hydroxyamide using the above-mentioned nitrile hydratase of thepresent invention.

Any patents, patent applications, and publications cited herein areincorporated by reference.

Herein, “%” for concentration denotes weight per volume percent unlessotherwise specified.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the determination of molecular weight of nitrile hydrataseof the present invention by SDS-PAGE. The abscissa indicates therelative mobility and the ordinate indicates the molecular weight (kDa).

FIG. 2 shows an elution profile of nitrile hydratase of the presentinvention by column chromatography with butyl-Toyopearl 650M. Whitesquare (□) represents OD₂₈₀, closed circle (●) represents the activityand hyphen (-) represents (NH₄)₂SO₄.

FIG. 3 shows the determination of molecular weight of nitrile hydrataseof the present invention by gel filtration. The abscissa indicatesretention time (minute) and the ordinate indicates the molecular weight(kDa).

FIG. 4 shows the Km value of nitrile hydratase of the present inventionfor HMBN, which was determined with an S-V plot.

FIG. 5 the Km value of nitrile hydratase of the present invention forHMBN, which was determined with a Lineweaver-Burk plot.

FIG. 6 shows the Km value of nitrile hydratase of the present inventionfor 3-cyanopyridine, which was determined with an S-V plot.

FIG. 7 shows the Km value of nitrile hydratase of the present inventionfor 3-cyanopyridine, which was determined with a Lineweaver-Burk plot.

FIG. 8 shows the effect of temperature on the activity of nitrilehydratase of the present invention.

FIG. 9 shows the thermal stability of nitrite hydratase of the presentinvention.

FIG. 10 shows the effect of pH on the activity of nitrile hydratase ofthe present invention.

FIG. 11 shows the pH stability of the nitrile hydratase of the presentinvention.

FIG. 12 shows the organization of gene cluster in Rhodococcus sp. Cr4.The nucleotide sequences indicated by capital letters correspond to theORFs. The names of subunits encoded the ORFs are shown at the right ofthe nucleotide sequences.

FIG. 13 shows the amino acid comparison between α-subunits of nitrilehydratase from Rhodococcus sp. Cr4 and Rhodococcus rhodochrous J1.Between Rhodococcus sp. Cr4 (top panel; CrNH-α) and Rhodococcusrhodochrous J1 (bottom panel; J1 L-a), identical amino acids arerepresented by an asterisk and distinct amino acids are represented by aspace.

FIG. 14 shows the amino acid comparison between β-subunits of nitrilehydratase from Rhodococcus sp. Cr4 and Rhodococcus rhodochrous J1.Between Rhodococcus sp. Cr4 (top panel; CrNH-β) and Rhodococcusrhodochrous J1 (bottom panel; J1 L-β), identical amino acids arerepresented by an asterisk and distinct amino acids are represented by aspace.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is lustrated in detail below with reference toExamples, but is not to be construed as being limited thereto.

1. Assay Method for the Enzymatic Activity

The standard assay method for the nitrile hydratase activity used in thefollowing Examples as follows. The standard composition of reactionsolution for the enzyme reaction is shown in Tables 1 and 2. The enzymereaction is initiated by adding 3-cyanopyridine or2-hydroxy-4-methylthiobutyronitrile (HMBN) as the substrate compound. Inthe case of 3-cyanopyridine, incubation was continued at 20° C. for 10minute, in the case of HMBN, at 20° C. for 15 minutes. When3-cyanopyridine was used in the reaction, 0.1 ml of 2 N hydrochloricacid was added to the reaction and the mixture was shaken vigorously tostop the reaction; when HMBN was used in the reaction, 0.1 ml of thereaction solution was added to 0.9 ml of 0.1% (v/v) phosphoric acid andthe mixture was shaken vigorously to stop the reaction. The reactionsolution was analyzed by HPLC.

TABLE 1 10% (v/v) HMBN in 0.1 M KPB (pH 6.5) 0.36 ml 0.1 M KPB (pH 6.5)0.64 ml Enzyme solution 0.10 ml 0.85% (w/v) NaClaq 0.90 ml Total volume2.00 ml

-   10% (v/v) HMBN was added to initiate the reaction.-   The mixture was incubated with shaking at 20° C. for 10 minutes.-   0.1% (v/v) H₃PO₄ was added to stop the reaction.-   Centrifugation.-   HPLC analysis.

TABLE 2 0.3 M 3-cyanopridine 1.00 ml 0.1 M KPB (pH 7.0) 0.50 ml Enzymesolution 0.10 ml 0.85% (w/v) NaClaq 0.90 ml Total volume 2.00 ml

-   0.3 M 3-cyanopyridine was added to initiate the reaction.-   The mixture was incubated with shaking at 20° C. for 3 minutes.-   2 N HCl was added to stop the reaction.-   Centrifugation.-   HPLC analysis.

The conditions used in HPLC analysis of the reaction solution: Theconditions used in HPLC analysis for HMBN are as follows:

Column: Spherisorb S5ODS2 (4.6 × 150 nm); Mobile phase: 0.1% (v/v)phosphoric acid/acetonitrile = 9/1; Flow rate: 1.0 ml/min.; Detection:UV 210 nm; Column temperature: 40° C.HPLC Analysis for the Enzymatic Activity:

The generated nicotinamide or HMBAm was quantified by HPLC, and thenitrile hydratase activity was calculated. The conditions of HPLC assayHMBAm were the same as that for HMBN. 1 U was defined as the enzymequantity capable of producing 1 μmol nicotinamide with the standardcomposition of reaction solution at 20° C. for 1 minute; or 1 U wasdefined as the enzyme quantity capable of producing 1 μmol HMBAm withthe standard composition of reaction solution at 20° C. for 1 minutes.

Protein Quantification:

The quantity of protein was determined according to Bradford method(Bradford, M., Anal, Biochem., 72, 248(1976)) with a protein assay kitfrom Bio-Rad.

Reagent:

DEAE-Sephacel and Butyl-Toyopearl 650 M used was provided by Pharmacia;bovine serum albumin, from Bio-Rad; molecular weight marker forSDS-PAGE, from Pharmacia; molecular weight marker for HPLC, fromOriental yeast. Unless otherwise specified, other reagents used werecommercially available special-grade reagents.

2. Culture Conditions

The pre-culture medium of the following composition was aliquoted in 5ml into each test tube (25×200 mm); a silicone plug was placed in thetube, followed by sterilization by autoclaving. After the tube wascooled, a bacterial strain was inoculated with a platinum loop, and thencultured with shaking at 28° C. for two days.

Pre-culture medium (pH 7.0): Polypeptone 5.0 g Meat extract 5.0 g NaCl2.0 g Yeast extract 0.5 g Distilled water 1.0 L

Then, the pre-culture was transferred into 20-ml main-culture mediumautoclaved in a 500-ml Sakaguchi flask. In the main culture, 0.75% (v/v)acetonitrile was added with a feeding needle after 24-hour culture, andthen the incubation was continued with shaking at 33° C. for two days.

Main-culture medium (pH 7.0): Acetamide 7.5 g Glucose 10.0 g C.S.L. 10.0g Yeast extract 1.0 g MgSO₄7H₂O 0.5 g K₂HPO₄ 1.0 g CoCl₂6H₂O 20.0 mgDistilled water 1.0 L3. Preparation of Cell-Free Extract

The bacterial cells corresponding to 500 ml of the culture liquid weresuspended at a 5-times higher cell density in 50 mM phosphate buffer/44mM n-butyric acid (pH 7.0). The suspended bacterial cells werehomogenized with a sonicator 201 M (Kubota) with 150 W at 4° C. or alower temperature for 60 minutes. Then, the culture was separated intothe supernatant and precipitating fractions by centrifuge at 13,000 rpmfor 20 minutes. The supernatant was used as cell-free extract for thefollowing purification.

4. Ammonium-Sulfate Fractionation

Ammonium sulfate was added to the cell-free extract at 30% saturation.The mixture was neutralized to pH 7.0 with 10%(v/v) ammonium water, andthen stirred at 4° C. for three hours. The solution was thenfractionated into the supernatant and precipitating fractions bycentrifuging at 13,000 rpm for 30 minutes. Ammonium sulfate was added tothe supernatant fraction at 60% saturation. The mixture was neutralizedto pH 7.0, and then stirred at 4° C. for three hours. The solution wasthen fractionated into the supernatant and precipitating fractions bycentrifuging. Further, ammonium sulfate was added to the supernatantfraction at 80% saturation. After stirred, the solution was fractionatedinto the supernatant and precipitating fractions. The respectiveprecipitates were suspended in 10 mM phosphate buffer/44 mMn-butyricacid (pH 7.0). The solutions were dialyzed three times against the samebuffer. Then the activity was assayed.

5. DEAE-Sephacel Column Chromatography

The enzyme solution, which had been sufficiently dialyzed against thesame buffer, loaded onto a column (φ13×216 mm) of DEAE-Sephacelsufficiently equilibrated with 10 mM phosphate buffer/44 mM n-butyricacid (pH 7.0). The column was then washed with the same buffer. Then,elution was carried out with the following elution buffer, and theenzymatic activity in the fractions was assessed. The nitrile hydrataseactivity was found in the fractions of No.74-80 when eluted with 10 mMphosphate buffer/44 mM n-butyric acid (pH 7.0)/0.3 M KCl. The volume ofeach elution buffer used was approximately 3 times as much as-the volumeof carrier.

-   10 mM phosphate buffer/44 mM n-butyric acid (pH 7.0)/0.1 M KCl-   10 mM phosphate buffer/44 mM n-butyric acid (pH 7.0)/0.2 M KCl-   10 mM phosphate buffer/44 mM n-butyric acid (pH 7.0)/0.3 M KCl-   10 mM phosphate buffer/44 mM n-butyric acid (pH 7.0)/0.4 M KCl    6. Column Chromatography with Butyl-Toyopearl 650 M

A column (φ11×89 mm) containing Butyl-Toyopearl was sufficientlyequilibrated with the buffer of 10 mM phosphate/44 mM n-butyric acid (pH7.0) containing ammonium sulfate at 20% saturation. Ammonium sulfate wasadded to the enzyme solution at 20% saturation, and the mixture wasstirred. The enzyme solution was loaded onto this column. The column waswashed with the same buffer. Then, elution was carried out with thefollowing elution buffer, and the enzymatic activity in the fractionswas assessed. The nitrile hydratase activity was found in the fractionsof No. 58-66 when eluted with 10 mM phosphate buffer/44 mM n-butyricacid (pH 7.0)/ammonium sulfate of 15% saturation. The volume of eachelution buffer used was approximately 3 times as much as the volume ofcarrier.

10 mM phosphate buffer/44 mM n-butyric acid (pH 7.0)/ammonium sulfate of15% saturation

10 mM phosphate buffer/44 mM n-butyric acid (pH 7.0)/ammonium sulfate of10% saturation

10 mM phosphate buffer/44 mM n-butyric acid (pH 7.0)/ammonium sulfateof. 5% saturation

7. SDS-PAGE

The purified enzyme obtained was analyzed by SDS-PAGE. SDS-PAGE wascarried out according to Laemmli's method (Laemmli, U.K.: Nature, 227,pp680, 1970). Namely, electrophoresis was performed in a 12%polyacrylamide slab gel with Tris/glycine buffer. An equal-volumemixture of enzyme solution and sample buffer was heat-treated at 90° C.for about 10 minutes. The gel was stained with Coomassie brilliant blueR-250; destaining was carried out with ethanol/acetic acid/dH₂O (2/3/6,by vol.). The result was shown in FIG. 1. The purified enzyme wasrevealed to consist of α-subunit (26.8 kDa) and β-subunit (29.5 kDa).

8. Gel Filtration by HPLC

The nitrile hydratase was purified according to the method as describedabove. First, the suspension of homogenized bacterial cells wasfractionated into the supernatant and precipitating fractions bycentrifugation. The activity was contained in the supernatant fraction,and thus the supernatant was used as a cell-free extract. The cell-freeextract was fractionated with ammonium sulfate, and, as a result, theactivity was contained in the fractions of which ammonium sulfateconcentration is 30-60% saturation. The active fractions were subjectedto column chromatography with DEAE-Sephacel. The active fractions (No.74-80) eluted from the column of DEAE-Sephacel were collected, and thensubjected to column chromatography with Butyl-Toyopearl 650 M. Theactive fractions (No. 0.58-66) eluted from the column of Butyl-Toyopearl650 M were used as the purified enzyme (FIG. 2).

Finally, with respect to the nitrile hydratase of Rhodococcus sp. Cr4,the total protein content was 5.32 mg; specific activity, 477 U/mg.;yield, 44%; specific activity, 13.9 times (Table 3). The purified enzymewas assessed to be homogeneous by SDS-PAGE. The specific activity offinally purified enzyme was 880 U/mg for HMBN and 477 U/mg for3-cyanopyridine used as the substrate under the standard conditions.

TABLE 3 Total Total Specific Purifi- protein activity activity cationYield Step (mg) (U) (U/mg) (-fold) (%) 1. Cell-free extract 167 574034.4 1 100 2. (NH4)2SO4 fractionation 56.6 5220 92.2 2.68 91 3.DEAE-Sephacel 18.7 4030 215 6.25 70 4. Butyl-Toyopearl 650 M 5.32 2540477 13.9 441 U of the enzyme is defined as the amount to catalyze the production of1 μmol nicotinamide from 3-cyanopyridine under the standard conditionsduring 1 minute.

About 2.3 μg of purified enzyme was analyzed by gel filtration under theconditions as shown in Table 4 to determine the molecular weight; themolecular weight of the enzyme in the natural state was deduced to beabout 112.5 kDa, when it was calculated based on the retention time forthe molecular weight marker (FIG. 3).

Gel Filtration by HPLC:

The conditions used for the HPLC analysis of nitrile hydratase for themolecular weight is shown in Table 4. The molecular weight marker usedwas from Oriental Yeast.

TABLE 4 Column: TSK gel G-3000 SW (0.75 × 60 cm) Solvent: 0.1 M KPB (pH7.5) + 0.2 M KCl Flow rate: 0.7 ml/min Injection: 5 μl Detection: 280 nm9. The Effect of Substrate Concentration (Km Value)

The Km value for HMBN was determined from the S-V plot (FIG. 4) andLineweaver-Burk plot (FIG. 5). 0.0585 U of the enzyme was added tosolutions containing HMBN at various concentrations and the reactionswere incubated at 20° C. for 15 minutes. The reactions were analyzed byHPLC. The Km value for HMBN was determined to be 1.43 mM from theLineweaver-Burk plot, and this indicates that the enzyme has highaffinity for HMBN.

In addition, the Km value for 3-cyanopyridine was determined from theS-V plot (FIG. 6) and Lineweaver-Burk plot (FIG. 7). Similarly, 0.0265 Uof the enzyme was added to solutions containing 3-cyanopyridine atvarious concentrations and the reactions were incubated at 20° C. for 15minutes. The reactions were analyzed by HPLC. The Km value for3-cyanopyridine was determined to be 1.38 mM from the Lineweaver-Burkplot, and this indicates that the enzyme also has high affinity for3-cyanopyridine.

10. The Effect of Temperature

The optimal temperature and thermal stability of the purified enzyme wasevaluated. With the standard composition of reaction solution as shownin Table 1, 2.92 U enzyme was incubated at each temperature (10, 20, 30,35, 40, 45, 50, 55, 60, or 65° C.) for 15 minutes to assess the optimaltemperature (FIG. 8). The temperature, at which the reaction rate withthe enzyme was maximized, was 45° C. Further, 2.92 U of the enzyme washeat-treated at each temperature (10, 20, 30, 40, 50, or 60° C.) for 30minutes, and then the thermal stability was assessed under the standardreaction conditions. After the enzyme treated at 50° C., the remainingactivity was 77% (FIG. 9).

11. The Effect of pH

The optimal pH and pH stability of the purified enzyme was evaluated.With the standard composition of reaction solution as shown in Table 1,2.92 U enzyme was incubated in each buffer (buffer of sodium citrate orTris-HCl at a final concentration of 50 mM), instead of phosphatebuffer, at 20° C. for 15 minutes to determine the optimal pH. Theoptimal pH was 6.0 (FIG. 10).

Further, 2.92 U enzyme was incubated in each buffer (buffer of sodiumcitrate, phosphate buffer, Tris-HCl, Glycine/NaOH or Na₂HPO₄/NaOH at afinal concentration of 50 mM) at 20° C. for 30 minutes, and then theremaining activity was assayed under the standard reaction conditions toassess the pH stability. After the enzyme treated at pH 4.0-9.0 for 30minutes, the remaining activity was nearly 100% (FIG. 11).

12. Substrate specificity

The substrate specificity of the enzymewas examined. With the standardcomposition reaction solution shown in table 2, 2.92 U enzyme wasincubated with various types of substrates, instead of 3-cyanopyridine;the generation of amides in the reaction solution was analyzed by HPLC.Thus, the relative activity was determined. The types of substratecompounds added, final concentrations thereof, reaction times, and HPLCconditions used for the analyses of products corresponding to therespective substrates are shown in Table 6. The HPLC conditions (1)-(9)are indicated next to Table 6.

The purified nitrile hydratase showed substrate specificity that wasvery similar to that of resting cell reaction; malononitrile,n-butyronitrile and 2-cyanopyridine were suitable substrates. Therelative activity for HMBN was 154%, when the activity for3-cyanopyridine was taken as 100% (Table 5).

TABLE 5 Relative activity Substrate (%) 3-Cyanopyridine 1002-Cyanopyridine 223 4-Cyanopyridine 127 Acrylonitrile 114Methacrylonitrile 93.7 Crotononitrile 87.4 Acetonitrile 14.6Propionitrile 173 HMBN 154 KCN 0 Malononitrile 673 6.16 2-Cyanoacetamide46.3 Cyanopyrazine 114 3-Cyanopyridine* 76.5 n-Butyronitrile* 379Isobutyronitrile* 40.9 n-Valeronitrile* 118 Isovaleronitrile* 2.71n-Capronitrile* 18.1 3-Indolylacetonitrile* 2.33 Benzonitrile* 138o-Chlorobenzonitrile* 58.6 m-Chlorobenzonitrile* 7.18p-Chlorobenzonitrile* 28.4The reaction was carried out at 20° C., and the amount of enzyme usedwas 2.92 U. The asterisk “*” indicates that methanol was added toenhance the substrate solubility (final conc.=20% v/v).

TABLE 6 Final Reaction concentration time Substrate (mM) (min) HPLCconditions 3-Cyanopyridine 150 15 (1) 3-Cyanopyridine* 150 15 (1)2-Cyanopyridine 125 10 (1) 4-Cyanopyridine 125 10 (1) Acrylonitrile 2503 (5) Methacrylonitrile 150 5 (1) Crotononitrile 150 5 (1) Acetonitrile250 10 (4) Propionitrile 150 5 (4) n-Butyronitrile* 150 10 (2)Benzonitrile* 50 10 (3) o-Chlorobenzonitrile* 25 5 (3)m-Chlorobenzonitrile* 25 5 (3) p-Chlorobenzonitrile* 25 5 (3)Malononitrile 250 3 (6) Isobutyronitrile* 150 10 (7) n-Valeronitrile*150 10 (7) Isovaleronitrile* 150 10 (7) n-Capronitrile* 150 10 (9)Cyanopyrazine 150 10 (8) 3-Indolylacetonitrile* 50 5 (9)The conditions for HPLC were as follows:

-   (1) Column: Waters Spherisorb 5 μODS2 (4.6×150 mm);-   Solvent: 10 mM KH₂PO₄(pH 2.8)/acetonitrile (v/v)=9:1;-   Flow rate: 1.0 ml/min;-   Detection: 230 nm;-   Injection volume: 5 μl.-   (2) Column: Waters Spherisorb 5 μODS2 (4.6×150 mm);-   Solvent: 10 mM KH₂PO₄(pH 2.8)/acetonitrile (v/v)=9:1;-   Flow rate: 1.0 ml/min;-   Detection: 230 nm;-   Injection volume:5 μl.-   (3) Column: Waters Spherisorb 5 μODS2 (4.6×150 mm);-   Solvent: 5 mM KH₂PO₄ (pH 2.8)/acetonitrile (v/v)=12:7;-   Flow rate: 1.0 ml/min;-   Detection: 230 nm;-   Injection volume: 5 μl.-   (4) Column: Waters Spherisorb 5 μODS2 (4.6×150 mm);-   Solvent: 10 mM KH₂PO₄(pH 2.5)/acetonitrile (v/v)=99:1;-   Flow rate: 1.0 ml/min;-   Detection: 210 nm;-   Injection volume: 5 μl.-   (5) Column: Waters Spherisorb 5 μODS2 (4.6×150 mm);-   Solvent: 10 mM KH₂PO₄(pH 2.5)/acetonitrile (v/v)=99:1;-   Flow rate: 1.0 ml/min;-   Detection: 230 nm;-   Injection volume: 5 μl.-   (6) Column: Spherisorb S5ODS2 (4.6×150 mm)-   Solvent: 0.1% (v/v) phosphate/acetonitrile (v/v)=99:1;-   Flow rate: 1.0 ml/min;-   Detection: 210 nm;-   Injection volume: 5 μl.-   (7) Column: Spherisorb S5ODS2 (4.6×150 mm);-   Solvent: 0.1% (v/v) phosphate/acetonitrile (v/v)=9:1;-   Flow rate: 1.0 ml/min;-   Detection: 210 nm;-   Injection volume: 5 μl;-   Temperature: 40° C.-   (8) Column: Spherisorb S5ODS2 (4.6×150 mm);-   Solvent: 0.1%(v/v) phosphate/acetonitrile (v/v)=9:1;-   Flow rate: 1.0 ml/min;-   Detection: 230 nm;-   Injection volume: 5 μl-   Temperature: 40° C.-   (9) Column: Spherisorb S5ODS2 (4.6×150 mm);-   Solvent: 5 mM KH₂PO₄ (pH 2.8)/acetonitrile (v/v)=12:7;-   Flow rate: 1.0 ml/min;-   Detection: 210 nm;-   Injection volume: 5 μl;-   Temperature: 40° C.    13. The Effect of Inhibitors on the Enzymatic Activity

Inhibitors to the enzyme were studied. With the standard composition ofreaction solution shown in Table 1, various inhibitors were added at afinal concentration 1.0 mM or 0.1 mM to the solutions containing 2.92 Uenzyme; after the mixtures were incubated at 20° C. for 10 minutes, thereaction was carried out for 15 minutes. The enzyme activity wasmarkedly inhibited by carbonyl reagents such as phenyl hydrazine andhydroxylamine (Table 7).

TABLE 7 The effects of various compounds on the nitrile hydrataseactivity Compound (1.0 mM) Relative activity (%) Free 100 Iodoacetate97.0 N-Ethylmaleimide 101 p-Chloromercuribenzoate 97.7 5-5′-Dithiobis(0.1 mM) 99.1 Hydroxylamine 62.1 Phenylhydrazine 8.19 Cysteamine 106D-Cycloserine 94.8 EDTA 101 Tiron 132 Diethyldithiocarbamate 97.7 Urea95.7 NaN₃ 98.5 Dithiothreitol 93.4 The enzyme was incubated with variouscompounds at 20° C. for 20 minutes, and the enzymatic activity wasassayed.14. The Effect of Metal Ion on the Enzymatic Activity

The effect of metal ion the enzyme reaction was studied. With thestandard composition of reaction solution shown in Table 1, variousmetal ions were added at a final concentration 1.0 mM to the solutionscontaining 2.92 U enzyme; after the mixtures were incubated at 20° C.for 10 minutes, the reaction was carried out for 15 minutes. The enzymeactivity was markedly inhibited by heavy metal ions such as Ag⁺ and Hg⁺⁺ions capable of specifically interacting with SH groups.

On the contrary, the addition of Ni⁺⁺ or Co⁺⁺ ion enhanced the enzymaticactivity (Table 8).

TABLE 8 The effect of metal ion on the nitrile hydratase activity Metal(1.0 mM) Relative activity (%) Free 100 CaCl₂ 96.4 MnCl₂ 96.7 NiCl₂ 196CoCl₂ 154 ZnSO₄ 84.1 CuSO₄ 109 FeSO₄ 78.9 FeCl₃ 100 AgNO₃ 7.85 HgCl₂7.48The enzyme was incubated with a metal ion at 20° C. for 10 minutes, andthe enzymatic activity was assayed.15. The Effect of Coexisting Ni⁺⁺ and Co⁺⁺ Ions on the EnzymaticActivity

It was revealed that the addition of Ni⁺⁺ or Co⁺⁺ ion to the reactionsystem enhanced the enzymatic activity. With the standard composition ofreaction solution shown in Table 1, Ni⁺⁺ or Co⁺⁺ ion was added at aconcentration of 0-8.0 mM to a solution containing 2.92 U enzyme; itseffect on the enzymatic activity was studied. When Ni⁺⁺ or Co⁺⁺ ion wasadded at a concentration of 1.0-2.0 mM, the enzymatic activity wasenhanced twice or 1.5 times. As the metal ions were added at higherconcentration than above-mentioned one, the enhancement was impaired(Tables 9 and 10).

TABLE 9 The effect of cobalt ion on the nitrile hydratase activity CoCl₂(mM) Relative activity (%) 0 100 0.5 129 1.0 150 2.0 153 4.0 143 6.0 1368.0 127The enzyme incubated with nickel ion at 20° C. for five minutes, and theenzymatic activity was assayed.

TABLE 10 NiCl₂ (mM) Relative activity (%) 0 100 0.5 171 1.0 197 2.0 1964.0 195 6.0 184 8.0 185

The enzyme incubated with nickel ion at 20° C. for five minutes, and theenzymatic activity was assayed.

16. The Effect of Coexisting Ni⁺⁺ Ion

As shown in Table 10, the enzymatic activity of nitrile hydratase of thepresent invention was enhanced twice by adding Ni⁺⁺ ion to the reactionsystem at a concentration of 1.0 mM. Then, with the standard compositionof reaction solution shown in table 2, various types of substrates,instead of 3-cyanopyridine, were added to the solutions containing 2.92U enzyme to study their effects on the activity. The activity wasenhanced twice only when HMBN or mandelonitrile was used as thesubstrate. The addition of Ni⁺⁺ ion was assumed to specifically enhancethe reaction efficiency when the substrate was a α-hydroxynitrile (Table11).

TABLE 11 Substrate Free NiCl₂ (1 mM) HMBN 100 196 3-cyanopyridine 100 992-cyanopyridine 100 97 4-cyanopyridine 100 99 n-butylonitrile 100 94Chlotononitilile 100 103 Manderonitlile 100 201 Etylene cyanohydrin 10091 Aminoacetonitlile 0 0 Hydroxyacetonitlile 0 0 3-aminopropyonitlile100 97 β-aminochlotononitlile 0 0The incubation was prolonged for 10 minutes at 20° C. using 2.92 Uenzyme.17. Determination of N-Terminal Amino Acid Sequence

The N-terminal amino acid sequences of α-subunit and β-subunit ofnitrile hydratase from Rhodococcus sp. Cr4 strain were analyzed in aProcise Sequencer Model 470A from Applied Biosystem. The sequence ofN-terminal 15 residues of α-subunit was TAHNPVQGTFPRSNE (SEQ ID NO: 5);that of β-subunit was MDGIHDLGGRAGLGP (SEQ ID NO: 6).

The respective sequences exhibited 93% identity to the sequence ofN-terminal 15 residues of α-subunit and 100% identity to that ofα-subunit of the low molecular weight nitrile hydratase from Rhodococcusrhodochrous J1 strain (Table 12/α-subunit; Table 13/β-subunit).

TABLE 12 Strain N-terminal amino acid sequence Identity (%) Rhodococcussp. Cr4  1 TAHNPVQGTFPRSNE 15 — Rhodococcus  1 TAHNPVQGTLPRSNE 15 93rhodochrous J1 (low) Bacillus subtilis 211 VVSNPLAGSRPRSND 225 47

TABLE 13 Identity Strain N-terminal amino acid sequence (%) Rhodococcussp. Cr4 1 MDGIHDLGGRAGLGP 15 — Rhodococcus rhodochrous 1 MDGIHDLGGRAGLGP15 100  J1 (low) Rhodococcus rhodochrous 1 MDGIHDTGGMTGYGP 15 73 J1(high) Pseudomonas putida 1 MNGIHDTGGAHGYGP 15 67 Pseudomonaschlororaphis 1 MDGFHDLGGFQGFGK 15 67 B2319. The Effect of Coexisting NiCl₂ and CoCl₂ in Resting Cell Reaction

The preculture medium of the following composition was aliquoted in 5 mlinto each test tube (25×200 mm); a silicone plug was placed in the tube,followed by sterilization by autoclaving.

Pre-culture medium (pH 7.0): Polypeptone 5.0 g Meat extract 5.0 g NaCl2.0 g Yeast extract 0.5 g Distilled water 1.0 L

After the tube was cooled, Rhodococcus sp. Cr4 or Rhodococcusrhodochrous ATCC 332878 strain was inoculated with a platinum loop intothe pre-culture medium, and then cultured with shaking at 28° C. for twodays. Then, the pre-culture of Rhodococcus sp. Cr4 was transferred intomain-culture medium 1; that of Rhodococcus rhodochrous ATCC 332878 wastransferred into main-culture medium 2. Both were cultured with shakingat 33° C. for two days. The bacterial cells were harvested and washed,and then the reaction shown in Table 14 was carried out to study theeffect of NiCl₂ and CoCl₂ added.

When NiCl₂ or CoCl₂ was added to the reaction system at a finalconcentration of 1 mM, as seen Table 15, the coexisting NiCl₂ or CoCl₂enhanced the nitrile hydratase activity, and thus the addition of theions had positive effect.

Main-Culture Medium 1 (for Rhodococcus sp. Cr4) (pH 7.0):

Acetamide 7.5 g Glucose 10.0 g C.S.L. 10.0 g Yeast extract 1.0 gMgSO₄7H₂O 0.5 g K₂HPO₄ 1.0 g CoCl₂6H₂O 10.0 mg FeSO₄7H₂O 10.0 mgDistilled water 1.0 LMain-Culture Medium 2 (for Rhodococcus rhodochrous ATCC 332878) (pH7.0):

ε-caprolactam 5.0 g Glucose 10.0 g C.S.L. 10.0 g Yeast extract 1.0 gMgSO₄7H₂O 0.5 g K₂HPO₄ 1.0 g CoCl₂6H₂O 20.0 mg Distilled water 1.0 L

Each medium was aliquoted in 20 ml into 500-ml Sakaguchi flasks, andthen used after sterilized by autoclaving.

TABLE 14 10% (v/v) HMBN in 0.1 M KPB (pH 6.5) 0.36 ml 0.1 M KPB (pH 6.5)0.64 ml Cell suspended solution (final: 0.05 fold) 0.10 ml 0.85% (w/v)NaClaq 0.90, 0.80 ml 20 mM NiCl₂ or CoCl₂ 0.00, 0.10 ml Total volume2.00 ml

-   HMBN was added (reaction started).-   The mixture was incubated with shaking at 20° C. for 15 minutes.-   0.1%(v/v) H₃PO₄ was added (the reaction stopped).-   Centrifugation.-   HPLC analysis.

TABLE 15 Relative activity (%) Rhodococcus sp. Cr4 ATCC 332878 Free 100*  100** NiCl₂ 186 193 CoCl₂ 137 123 *118 μmol/ml/min **30.5μmol/ml/minIn resting cell reaction, the enzymatic activity was also enhanced byNiCl₂ or CoCl₂ added.20. The Effect of Cyanide Ion

The effect of cyanide ion on the nitrile hydratase of the presentinvention derived form Rhodococcus sp. Cr4, and known nitrile hydratasederived from Rhodococcus rhodochrous J1 (Biochimica et Biophysica Acta.1129 (1991): 23-33) was studied. With the following standard compositionof reaction solution, 0 mM-20 mM cyanide ion (KCN) was added to thereaction system. After the reaction solution was incubated in theabsence of substrate (3-cyanopyridine) at 20° C. for 30 minutes, thesubstrate was added thereto to start the enzyme reaction. After theenzyme reaction at 20° C. for 10 minutes, 0.1 ml of 2 N hydrochloricacid was added thereto and the mixture was shaken vigorously to stop thereaction. The reaction solution was analyzed by HPLC by the same methodas described in Section 1.

Standard Reaction Solution:

0.5 M 3-cyanopyridine 0.5 ml 0.1 M phosphate buffer (pH 7.5) 0.25 ml Enzyme solution 0.1 ml Total volume 1.0 ml

In the case of Rhodococcus sp. Cr4 nitrile hydratase, the quantity ofenzyme used was 288 U/ml; in the case of Rhodococcus rhodochrous J1nitrile hydratase, the quantity of enzyme used was 61 U/ml. As seen inTable 16, the nitrile hydratase of Rhodococcus rhodochrous J1 wascompletely inhibited in the presence of 1 mM cyanide ion, but thenitrile hydratase of Rhodococcus sp. Cr4 retained 47% of the activity inthe presence of 1 mM cyanide ion and 17% of the activity in the presenceof 5 mM cyanide ion.

TABLE 16 KCN (mM) Cr4 J1 0 100% 100% 1 47 0 5 17 0 10 11 0 15 8 0 20 3 021. Determination of N-Terminal Amino Acid Sequence

The nitrile hydratase purified from Rhodococcus sp. Cr4 was subjected toSDS-PAGE. The protein was electro-transferred onto a PVDF membrane witha Horiz-Blot Electrophoresis Apparatus Model AE6678-P (ATTO), and thenstained with amide black. Portions corresponding to α-subunit andβ-subunit were cut off, and each of the subunit proteins were subjectedto automatic Edman degradation in a gas-phase peptide sequencer model473A (Applied Biosystem), and thus PTH amino acid derivatives wereobtained. The N-terminal amino acid sequences of α-subunit and β-subunitwere analyzed in a PTH amino acid derivative analyzer model 120A(Applied Biosystem).

The N-terminal sequence was TAHNPVQGTFPRSNE (SEQ ID NO: 5) forα-subunit; MDGIHDLGGRAGLGPI (SEQ ID NO: 7) for β-subunit. The sequenceof α-subunit exhibited 93% identity (L at amino acid residue 10 in J1)to the N-terminal sequence of α-subunit of low-molecular-weight nitrilehydratase from Rhodococcus rhodochrous J1; the sequence of β-subunit did100% identity to that of the equivalent from Rhodococcus rhodochrous J1.

Thus, the N-terminal amino acid sequences of nitrile hydratase α-subunitand β-subunit from Rhodococcus rhodochrous J1 are nearly identical tothose from Rhodococcus sp. Cr4. Further, it is known that α-subunits ofpreviously reported nitrile hydratases share the highly homologousprimary structures. Thus, cloning of the nitrile hydratase gene ofRhodococcus sp. Cr4 was carried out using, as a probe DNA, a genefragment from α-subunit of nitrile hydratase of Rhodococcus rhodochrousJ1. Further, a gene fragment form β-subunit of nitrile hydratase ofRhodococcus rhodochrous J1 was used to evaluate whether the entireregion of nitrile hydratase gene of Rhodococcus sp. Cr4 was successfullycloned.

22. Preparation of Probe DNA

There two types of nitrile hydratases in Rhodococcus rhodochrous J1,namely, the low molecular weight form and high molecular weight form.Therefore, firstly, regions of the primary structure of low molecularweight nitrile hydratase gene, of which sequences are different fromthose corresponding regions of the high molecular weight form, inRhodococcus rhodochrous J1, were selected. Specifically, the respectiveamino acid sequences selected are described below.

α-Subunit:

-   Sense primer region: QGTLPRSN (SEQ ID NO: 8)-   Antisense primer region: PDPDVEIR (SEQ ID NO: 9)    β-Subunit:-   Sense primer region:. PHDYLTSQ (SEQ ID NO: 10)-   Antisense primer region: PNVVNHID (SEQ ID NO: 11)

Then, based on the amino acid sequences, PCR primers, to be used forspecifically amplifying a part of the low molecular weight nitrilehydratase gene, were actually designed. Finally, the primers comprisingthe following nucleotide sequences were designed.

α-Subunit Gene Fragment for Amplification:

-   sense primer: 5′-cagggcacgttgccacgatcg-3′ (SEQ ID NO: 12)-   antisense primer: 5′-cggatctcgacgtcagggtcg-3′ (SEQ ID NO: 13)    β-Subunit Gene Fragment for Amplification:-   sense primer: 5′-cgcacgactacctgacctcgc-3′ (SEQ ID NO: 14)-   antisense primer: 5′-cgatgtgattgactacgttcgg-3′ (SEQ ID NO: 15)

Then, the genomic DNA was prepared from Rhodococcus rhodochrous J1according to the method of Ausbel et al. (Ausbel, F M et al: UNIT 2.4,Preparation of Genomic DNA from Bacteria in Current Protocols inMolecular Biology (John Wiley and Sons, New York) 1987). Further, thegenomic DNA was also prepared from culture cells of Rhodococcus sp. Cr4by the same method.

A part of the low molecular weight nitrile hydratase gene of Rhodococcusrhodochrous J1 was amplified by PCR using Taq DNA 30 (Takara Shuzo) andcycles of: denaturation at 94° C. for 1 minutes, annealing at 60° C. for90 seconds, and extension at 72° C. for 90 seconds using 10 ng of thegenomic DNA obtained as a template. Amplified DNA fragment was subjectedto agarose gel electrophoresis and then collected from the gel by usinga RECOCHIP (Takara Shuzo).

PCR amplification using the primers for the amplification of α-subunitgene fragment and the genomic DNAs from Rhodococcus rhodochrous J1 andRhodococcus sp. Cr4 as templates gave amplification products of about450 bp from both strains. However, when the genomic DNA from Rhodococcussp. Cr4 was used as the template, the efficiency of PCR amplificationwas lower. Thus, the amplified fragment by using the genomic DNA ofRhodococcus rhodochrous J1 as the template was used as the probe DNA forthe subsequent experiments.

The amplified DNA fragment derived from Rhodococcus rhodochrous J1 waslabeled with digoxigenin according to the random priming method with aDIG-labeling kit (Boehringer Manheim). The digoxigenin-labeled DNAfragment was used as the probe DNA in the subsequent Southern blotanalysis and colony hybridization.

23. Southern Blot Analysis

The genomic DNA of Rhodococcus sp. Cr4 was digested with variousrestriction enzymes, and analyzed by Southern blotting using the probeDNA from α-subunit of nitrile hydratase derived from Rhodococcusrhodochrous J1. 10 μg of the genomic DNA from Rhodococcus sp. Cr4 wascompletely digested with PstI, SphI and BanIII (Toyobo), and then 1-μgaliquots of the digests were electrophoresed on a 0.8% agarose gel. TheDNA was transferred onto a nylon membrane NY13N (Schleicher & Schuel) ina vacuum blotter model 785 (Bio-Rad) according to the attached manual.The DNA was immobilized on the membrane in an autocrosslinker CL-1000(BM Equipment) The resulting membrane was hybridized with the probe DNAat 60° C. overnight, and then washed at room temperature for 5 minutesand then at 60° C. for 15 minutes. The chemical luminescent light byCDP-Star was exposed onto a Fuji RX film for signal detection by using aDIG detection kit (Boehringer Manheim) according to the attached manual.

The analysis of Southern blotting revealed single bands of 3.2 kb, 4.2kb and 7.8 kb in the lanes of the genomic DNA from Rhodococcus sp. Cr4digested with PstI, SphI and BanIII, respectively. The nitrile hydratasegene of Rhodococcus sp. Cr4 was found to be a single-copy gene. Thenitrile hydratase gene of Rhodococcus sp. Cr4 was cloned from a genomiclibrary prepared from the PstI digest.

24. Preparation of a Genomic Library of Rhodococcus sp. Cr4 and ColonyHybridization

A plasmid pBluescript II SK (+) (Stratagene) was digested with PstI, andthen dephosphorylated by bacterial alkaline phosphatase (Takara Shuzo).The genomic DNA of Rhodococcus sp. Cr4 was digested with PstI, and thenligated to the above pBluescript II SK (+) by using T4 DNA ligase(Takara Shuzo). Then, E. coli DH5α was transformed by above-mentionedvector.

Colony hybridization was performed according to the method of Sambrooket al. (Molecular cloning (Cold Spring Harbor Laboratory Press, ColdSpring Harbor) 1989). Specifically, a nitrocellulose filter was placedon an LB agar plate containing 50 μg/ml ampicillin, and thetransformants obtained were spread thereon. The plate was incubated at37° C. overnight. After the colonies were formed, a replica filterprepared was placed on the agar plate and cultured thereon. After thecolonies were formed on the replica filter., the filter was transferredonto an agar plate containing 200 μg/ml chloramphenicol. The plate wasincubated at 37° C. for 24 hours. The filter was treated with SDS andalkali to lyse the colonies. Then, colony hybridization was carried outunder the same conditions as used in the analysis of Southern blotting.Thus, a positive clone was obtained from about 2000 colonies.

25. Analyses of the Positive Clone

After the positive clone was cultured, the plasmid was prepared by thealkali-SDS method; the plasmid was named pNHCr4P. The plasmid PNHCr4Pcontained an insert of about 3.2-kb DNA fragment.

In order to confirm that the insert DNA fragment of pNHCr4P contains theβ-subunit gene, a probe DNA was prepared by PCR using the primers forthe amplification of the β-subunit gene fragment and the genomic DNA ofRhodococcus rhodochrous J1 as a template. With this probe DNA, pNHCr4Pwas analyzed by Southern blotting. As a result, the insert fragment ofabout 3.2 kb containing the β-subunit gene was confirmed.

The nucleotide sequence of insert DNA fragment (about 3.2 kb) in pNHCr4Pwas analyzed by the dideoxy chain termination method using a DNAsequencer model 377A (Applied Biosystem) and Taq primer cycle sequencingkit (Applied Biosystem). The determined nucleotide sequence is shown inSEQ ID NO: 16. The sequencing result showed that the size of PstI insertDNA fragment in pNHCr4P was 3205 bp and that an ORF was presence (FIG.12). The organization of gene cluster in Rhodococcus sp. Cr4 was verysimilar to the region containing the low molecular weight nitrilehydratase gene in Rhodococcus rhodochrous J1 (Komeda et al: Proc NatlAcad Sci USA 94: 36-41, 1997).

26. Analysis of Primary Structure

The primary structures of α-subunit and β-subunit deduced from theregion of nucleotides 245-2100 of the PstI fragment are shown in SEQ IDNO: 2 and SEQ ID NO: 4, respectively. The N-terminal amino acidsequences of α-subunit and β-subunit completely agreed with thosedetermined with the protein sequencer. In addition, both α-subunit andβ-subunit exhibited the most-homologies to those of nitrile hydratasefrom Rhodococcus rhodochrous J1 (Table 17).

TABLE 17 α β R. rhodochrous J1 (low molecule type) 92% 87% Rhodococcussp. 65% 45% Pseudomonas putida 59% 37% Bacillus sp. BR449 60% 38%Mesorhizobium loti 56% 38% Sinorhizobium meliloti 55% 39% Rhodococcussp. M8 52% 37% R. rhodochrous J1 (high molecule type) 52% 37% R.erythropolis 50% 35% Pseudomonas chlororaphis B23 50% 31% Brevibacteriumsp. R312 50% ? Patent No. W09504828 42% 34%

17 amino acid residues of 207 residues of α-subunits of nitrilehydratase were different between Rhodococcus sp. Cr4 (SEQ ID NO: 2) andRhodococcus rhodochrous J1 (SEQ ID NO: 18). Further, 29 amino acidresidues of 227 residues of β-subunit of nitrile hydratase weredifferent between Rhodococcus sp. Cr4 (SEQ ID NO: 4) and Rhodococcusrhodochrous J1 (SEQ ID NO: 19) (FIGS. 13 and 14).

INDUSTRIAL APPLICABILITY

The present invention provides a nitrile hydratase capable of producing2-hydroxy-4-methylthiobutyroamide from2-hydroxy-4-methylthiobutyronitrile as a substrate.2-hydroxy-4-methylthiobutyroamide is a useful compound as a feedadditive (a methionine substitute).

Not only the present invention provides the nitrile hydratase capable ofenzymatically producing 2-hydroxy-4-methylthiobutyroamide, but also theencoding gene was cloned in the present invention. The nitrile hydrataseof the present invention can be expressed at high levels in appropriatehost cells transformed with the gene encoding the nitrile hydrataseprovided by the present invention. Thus, the transformants themselvesthat can be obtained according to the present invention, or the enzymeprotein obtained from the transformants, are useful for enzymaticallyproducing 2-hydroxy-4-methylthiobutyroamide. Though many of knownnitrile hydratases produced by genetic recombination could not achievehigh enzymatic activity, the nitrile hydratase of the present inventionis excellent retaining the high activity even when it is a geneticrecombinant.

Further, the nitrile hydratase of the present invention retains the highenzymatic activity in the presence of cyanide or aldehyde. In a polarsolvent, α-hydroxynitrile as the substrate compound is decomposed tohydrocyanic acid and aldehyde. Hydrocyanic acid is converted to cyanide,which often reduces the enzymatic activity. Aldehyde also gives damagesto the protein and reduces the enzymatic activity. One of the reasonswhy known nitrile hydratases were not industrially applicable was thatthese cyanide and aldehyde reduces the enzymatic activity.

The hydratase of the present invention retains the enzymatic activityeven in the presence of cyanide or aldehyde. Therefore, α-hydroxynitrileproduced from aldehyde and hydrocyanic acid can be used as thesubstrate. Thus, the nitrile hydratase of the present invention isuseful for the method for producing amides using α-hydroxynitrile asstarting material.

1. An isolated protein complex consisting of an α-subunit protein and a β-subunit protein, wherein said protein complex is capable of converting: a nitrile group of a nitrile compound to an amide group; and wherein the α subunit protein is a protein selected from the group consisting of: (A) a protein comprising the amino acid sequence of SEQ ID NO: 2; (B) a protein comprising the amino acid sequence of SEQ ID NO: 2 in which one to ten amino acids are substituted, deleted, inserted and/or added; (C) a protein having 95% or higher identity to the amino acid sequence of SEQ ID NO: 2; (D) a protein encoded by a polynucleotide comprising the nucleotide sequence of SEQ ID NO:1; and (E) a protein encoded by a polynucleotide that hybridizes to the nucleotide sequence of SEQ ID NO:1 under stringent conditions of 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C.; and wherein the β subunit protein is a protein selected from the group consisting of: (a) a protein comprising the amino acid sequence of SEQ ID NO: 4; (b) a protein comprising the amino acid sequence of SEQ ID NO: 4 in which one to ten amino acids are substituted, deleted, inserted and/or added; (c) a protein having 95% or higher identity to the amino acid sequence of SEQ ID NO: 4; (d) a protein encoded by a polynucleotide comprising the nucleotide sequence of SEQ ID NO:3; and (e) a protein encoded by a polynucleotide that hybridizes to the nucleotide sequence of SEQ ID NO:3 under stringent conditions of 6×SSC at about 45° C., followed by one or washes in 0.2×SSC, 0.1% SDS at 65° C.
 2. The protein complex according to claim 1, wherein the α-subunit protein comprises the amino acid sequence of SEQ ID NO:
 2. 3. The protein complex according to claim 1, wherein the β-subunit protein comprises the amino acid sequence of SEQ ID NO:
 4. 4. An isolated protein selected from the group consisting of: (A) an α-subunit protein comprising the amino acid sequence of SEQ ID NO: 2; (B) an α-subunit protein comprising the amino acid sequence of SEQ ID NO: 2 in which one to ten amino acids are substituted, deleted, inserted and/or added, wherein said α-subunit protein is capable of forming a protein complex with any β-subunit protein defined below in subsections (F) to (J) and said protein complex is capable of converting a nitrile group of a nitrile compound to an amide group; (C) an α-subunit protein having 95% or higher identity to the amino acid sequence of SEQ ID NO: 2, wherein said α-subunit protein is capable of forming a protein complex with any β-subunit protein defined below in subsections (F) to (J) and said protein complex is capable of converting a nitrile group of a nitrile compound to an amide group; (D) an α-subunit protein encoded by a polynucleotide comprising the nucleotide sequence of SEQ ID NO:1; (E) an α-subunit protein encoded by a polynucleotide that hybridizes to the nucleotide sequence of SEQ ID NO:1 under stringent conditions of 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C., wherein said α-subunit protein is capable of forming a protein complex with any β-subunit protein defined below in subsections (F) to (J) and said protein complex is capable of converting a nitrile group of a nitrile compound to an amide group; (F) a β-subunit protein comprising the amino acid sequence of SEQ ID NO: 4; (G) a β-subunit protein comprising the amino acid sequence of SEQ ID NO: 4 in which one to ten amino acids are substituted, deleted, inserted and/or added, wherein said β-subunit protein is capable of forming a protein complex with any α-subunit protein defined above in subsections (A) to (E) and said protein complex is capable of converting a nitrile group of a nitrile compound to an amide group; (H) a β-subunit protein having 95% or higher identity to the amino acid sequence of SEQ ID NO: 4, wherein said β-subunit is capable of forming a protein complex with any α-subunit protein defined above in subsections (A) to (E) and said protein complex is capable of converting a nitrile group of a nitrile compound to an amide group; (I) a β-subunit protein encoded by a polynucleotide comprising the nucleotide sequence of SEQ ID NO:3; and (J) a β-subunit protein encoded by a polynucleotide that hybridizes to the nucleotide sequence of SEQ ID NO:3 under stringent conditions of 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C., wherein said β-subunit protein is capable of forming a protein complex with any α-subunit protein defined above in subsections (A) to (E) and said protein complex is capable of converting a nitrile group of a nitrile compound to an amide group.
 5. The protein complex according to claim 1, wherein the α-subunit protein comprises the amino acid sequence of SEQ ID NO:2 and the β-subunit protein comprises the amino acid sequence of SEQ ID NO:4.
 6. The protein complex according to claim 1, wherein the protein complex is derived from Rhodococcus sp. Cr4 strain that has been deposited under the accession numbed FERM BP-6596.
 7. The protein complex according to claim 1, wherein the protein complex is capable of converting 2-hydroxy-4-methylthiobutyronitrile to 2-hydroxy-4-methylthiobutyroamide. 